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THESIS

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This is to certify that the

dissertation entitled

Novel Approaches for the Immunoassay of
Fusarium Myootoxins

presented by

Sutikno

has been accepted towards fulfillment
of the requirements for

Envimmnental Toxicology

 

 

Dr. James J. Pestka

Major professor

 

July 18, 1995

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

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MSU I. An Affirmative ActkWEqual Opportunity lnstltulon
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”31.1

NOVEL APPROACHES FOR THE IMMUNOASSAY OF
FUSARIUM MYCOTOXINS

By

Sufikno

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1995

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ABSTRACT
NOVEL APPROACHES FOR THE IMMUNOASSAY OF
FUSARIUM MYCOTOXINS
By

Sutikno

The fumonisin and trichothecence mycotoxins are a group of secondary
metabolites produced by toxigenic strains of Fusan'um fungi. These mycotoxins
are commonly toxic to both human and animals and found in agricultural
products. The presence of these toxins in agricultural commodities are primarily
dictated by environmental and biologic factors. Since controlling these factors is
almost impossible, mycotoxin entry into human and animal foods is typically
prevented through detection and diversion. Although mycotoxin detection is
performed using conventional methods such as high performance liquid
chromatography (HPLC), immunoassay techniques such as enzyme-linked
immunosorbent assay (ELISA) are inexpensive, simple, rapid, specific, and
sensitive. In this thesis, several new approaches for the generation and
application of antibodies to Fusarium mycotoxins were explored. Firstly, mouse
monoclonal, rabbit and sheep polyclonal antibodies against fumonisin B1 (F81 )
were produced via a novel immunization procedure with keyhole limpet hemacyanin

as a protein carrier. These antibodies were used to improve the sensitivity and

malty of E
senstivlly and s
mnpettive die
this ELISA pro
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specificity of ELISAs that were previously developed in our laboratory. ELISA
sensitivity and specificity were much higher when F81 -sheep antisera were used in
competitive direct ELISA. FB1 detection in Fusan‘um culture, and corn products with
this ELISA provided approximately two fold higher F81 estimates than that with
HPLC. In addition, ELISAs had a strong positive relationship with HPLC. suggesting
that they are suitable for fumonisins screening from human and animal foods.
Secondly, attempts to generating high affinity and specificity antibodies against
deoxynivalenol (DON) by immunizing mice and rabbits with a variety of DON-bovine
serum albumin conjugates were made to improve sensitivity of previous DON
competitive ELISA . High titer antisera were produced but they could not be used in
DON competitive ELISAs. Finally, attempts were made to generate antibodies
smcific to the hichothecene—yeast ribosomal binding site by immunizing mice with
808 ribosomal subunits. Although all animals exhibit high antiserum titers for the
ribosomes, these antisera were not effective in a variety of ELISA configurations to

measure “total load” trichothecenes in foods.

To my parents and my grandparents for their moral support and prayer.
To my wife Zuli and my daughter, Wulan, for their support,

patience, kindness, and understanding.

l am g
advice. guide
this dissertat:
and Dr. L Pat
graduate gun?

I WiSh ti
Olivera for the
Dioduction of m

SPeCial i
daughter. Tlti
understanding c

Finally,
almarltum, my
ll‘sSplre me to

University and ,

 

 

ACKNOWLEDGMENTS

I am greatly grateful to my major advisor, Dr. James J. Pestka, for his
advice, guidance, assistance, and understanding throughout the development of
this dissertation. I am also indebted to Dr. John E. Linz, Dr. William Helferich,
and Dr. L. Patrick Hart for their invaluable suggestion and being members of my
graduate guidance committee.

I wish to thanks to Dr. Mohamed M. Abouzied and Dr. Juan I. Azcona-
Olivera for their invaluable suggestion and practical help especially during the
production of monoclonal antibodies.

Special acknowledgment goes to my wife, Siti Zulaikah Sutikno, and my
daughter, Titiek Wulandari Sutikno, for their patience, supports, and
understanding during the course of my graduate program.

Finally, my appreciation extends to my late father, bapak Soewito
almarhum, my grandmother, Eyang Musini, and my brothers and sisters who ‘
inspire me to study and work very hard during my stay in Michigan State

University and to be a better Moeslim.

LIST OF TAE
LIST OF FIG
INTRODUCI
PART I. RE

FUSA RIUL
Introduc:
Fumonisi
Tn’chothe

CONVENT
Cleanup .
Detection

IMMUNOAS
Antibody ‘
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PART II DEV
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ABSTRACT

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TABLE OF CONTENTS

LIST OF TABLES .............................................................................................. ix
LIST OF FIGURES ............................................................................................ xi
INTRODUCTION ................................................................................................. 1
PART I. REVIEW OF FUSARIUM-MYCOTOXIN IMMUNOASSAY ................... 4
FUSARIUM MYCOTOXINS ............................................................................. 5
Introduction ................................................................................................... 5
Fumonisins ................................................................................................... 9
Trichothecenes ........................................................................................... 14
CONVENTIONAL ANALYTICAL METHODS .................................................. 28
Cleanup of trichothecenes prior to analytical detection ................................ 28
Detection of trichothecenes ........................................................................ 30
IMMUNOASSAYS FOR FUSARIUM MYCOTOXINS ..................................... 38
Antibody production .................................................................................... 39
Mycotoxin immunoassay format .................................................................. 49
Application of mycotoxin immunoassays in foods ........................................ 54
Commercial mycotoxin immunoassay kits for foods .................................... 57

PART II DEVELOPMENT AND APPLICATION OF AN ELISA FOR
FUMONISIN B1 IN FUNGAL CULTURES, CORN AND CORN

PRODUCTS ....................................................................................... 61
ABSTRACT ................................................................................................... 62
INTRODUCTION ........................................................................................... 63

Background. ............................................................................................... 63
Analytical methods ...................................................................................... 66
Fumonisin antibodies .................................................................................. 68
Rationale. ................................................................................................... 69

vi

 

 

MATERIAL
Chemical
Conluga‘.

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Direct E“
Veratox‘:

Com-$3
HPLC.

Statistics

RESULTS
Rabbit p:
Mouse lr
Compaq
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CONCLU:

PARTIH r
r

ABSTR

INTRQ{
Natur
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DON
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Rat
ELJ

MATERIALS AND METHODS ........................................................................ 71

Chemical and reagents ............................................................................... 71
Conjugation of fumonisin 81 to protein ........................................................ 72
Rabbit immunization ................................................................................... 73
Hybridoma production ................................................................................. 74
Indirect ELISA ............................................................................................. 76
Direct ELISA ............................................................................................... 77
Veratox® .................................................................................................... 78
ELISAGRAM ............................................................................................... 79
Fusan'um cultures ....................................................................................... 8O
Com-sample extraction and clean up .......................................................... 81
HPLC .......................................................................................................... 82
Statistics ..................................................................................................... 84
RESULTS AND DISCUSSION ....................................................................... 85
Rabbit polyclonal antibodies ....................................................................... 85
Mouse immunization and monoclonal antibodies ........................................ 86
Comparison among different antibodies ...................................................... 87
Detection of FBt-like compounds ................................................................ 89
Detection of F81 in Fusan'um cultures ......................................................... 90
Detection of F B1 in corn and corn product .................................................. 91
CONCLUSION ............................................................................................... 98

PART III PRODUCTION OF DEOXYNIVALENOL (VOMITOXIN)

ANTIBODIES ................................................................................... 122
ABSTRACT ................................................................................................. 123
INTRODUCTION ......................................................................................... 124

Natural occurrence. .................................................................................. 124
Toxicity ..................................................................................................... 126
DON detection .......................................................................................... 129
Rationale .................................................................................................. 1 30
MATERIALS AND METHODS ....................................................................... 132
Chemical and reagents ............................................................................ '. 132
Derivation of DON ..................................................................................... 132
Conjugation of DON derivatives to protein .................................................. 134
Mouse immunization .................................................................................. 134
Rabbit immunization ................................................................................. 136
ELISA ....................................................................................................... 137

vii

 

RESULTS
DONder
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CONCLUS
PARTIV.

ABSTTLAC

NTRODI
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RESULT
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CONCLL

APPENDI)

APPENDlx
APPENDI):

RESULTS .............................................................................................

DON derivatization ...................................................................... I333
Mouse Immunization ............. 139
Rabbit immunization 14o
DISCUSSION ............................................................................ 141
DON denvatlzatlon '''''''''' 141
Antibody production 141
CONCLUSION .............................................................................................. 143

PART IV. PRODUCTION OF ANTIBODIES AGAINST TRICHOTHECENE-

YEAST RIBOSOMAL BINDING SITE ........................................... 148
ABSTRACT ...................................................... 149
INTRODUCTION ......................................................................................... 1 50

Trichothecene mycotoxins ........................................................................ 150
Ribosomal binding as a toxicity mechanism ................................................. 151
Rationale and objective .............................................................................. 154
MATERIALS AND METHODS ...................................................................... 156
Chemicals and reagents ............................................................................. 156
Yeast production ........................................................................................ 157
Ribosome preparation ................................................................................ 157
T-2 toxin-ribosome binding assay ............................................................... 159
Mouse immunization .................................................................................. 160
Indirect ELISA ........................................................................................... 161
RESULTS AND DISCUSSION ..................................................................... 163
Ribosome preparation ............................................................................... 163
Mice immunization ..................................................................................... 164
CONCLUSION .............................................................................................. 167
APPENDIX A: MEDIA FOR HYBRIDOMA PRODUCING MONOCLONAL
ANTIBODIES .......................................................................... 172
APPENDIX B: SAMPLE LISTS OF CORN AND CORN PRODUCTS ............... 175

APPENDIX C: IMMUNOLOGICAL ASSAYS FOR MY COTOXIN DETECTION . 179
LIST OF REFERENCES ........................................................................................ 188

viii

“av-fl W". I

Table 1.1.

Table 1.2.

Table 1.3. h

Table 1.4.

Table 1.5.

Table 1.6.

Table 1.7_

Table 2.1.

Table 2.2.

 

Table 1.1.

Table 1.2.
Table 1.3.

Table 1.4.

Table 1.5.

Table 1.6.
Table 1.7.

Table 2.1.

Table 2.2.

Table 2.3.

Table 2.4.

Table 2.5.

LIST OF TABLES

Types Of economic losses and costs associated with mycotoxin
contamination in foods and feeds (CAST, 1989) ................................ 8

Natural occurrence of fumonisins in cereals and cereal products 15

Natural occurrence of trichothecene deoxynivalenol (DON) and
nivalenol (NIV) in cereals and cereal products ................................ 22

Summary of trichothecene-induced toxicoses ................................. 26

Retention factor (Rf) values of some trichothecene mycotoxins
on TLC plates using silica gel as absorbent (adapted from
Ueno, 1980) .................................................................................... 32

Selected Fusarium mycotoxin immunoassays in foods .................... 55

Commercial Immunoassay Kits for Mycotoxins available as of
June 1993 (Pestka et al., 1995) ...................................................... 58

Sensitivity of Cl-and CD-ELISAS for F81 using monoclonal
antibodies prepared from ascites fluid ........................................... 106

Comparison of competitive F81 ELISA using mouse-monoclonal
antibodies prepared from ascites fluid, rabbit-and sheep-
polyclonal antibodies ..................................................................... 1 10

Cross reactivity of FB1 sheep antisera toward fumonisin
analogues. .................................................................................... 112

Comparison of FBt concentrations in Fusarium corn cultures
detected by HPLC and CD-ELISA methods ................................. 115

Comparison of FBI recoveries from spiked-com samples
containing FB1 of 100 to 3,000 uglg using raw methanolic
extracts and SAX-cleaned extracts as determined by HPLC,

Veratox® ', and CD-ELISA using F81 Sheep antisera (Neogen ’
Corp., Lansing, MI) ....................................................................... 116

ix

 

Table 2.6.

031.0 .

Table 2.7.

.‘T m

A

Table 2.8.

 

I0 0

 

Q3.

Table 3.1. 81

Table 5.1.

Table 5.2_ h

Table 5_ 3_

'7

Table 2.6. Comparisons of F 81 concentrations in 14 com-food samples by
HPLC, Veratox® ', and CD-ELISA using FBt sheep antisera
(Neogen Corp., Lansing, MI) and the ratios of Veratox® ' and
CD-ELISA to HPLC results ........................................................... 117

Table 2.7. Comparisons of F81 concentrations in 28 Italian feed samples
by HPLC, Veratox® ', and CD-ELISA using F81 sheep antisera
(Neogen Corp., Lansing, MI) and the ratios of Veratox® " and
CD-ELISA to HPLC results ........................................................... 118

Table 2.8. Comparisons of F B1 concentrations in fresh com 3 samples by
HPLC, Veratox® '. and CD-ELISA using F81 sheep antisera
(Neogen Corp., Lansing, MI) and the ratios of Veratox® b and

CD-ELISA to HPLC results ........................................................... 120
Table 3.1. Structures of deoxynivalenol (DON) analogs (Adapted from

Ueno, 1983) .................................................................................. 125
Table 5.1. Number and description of food samples bought in retail

supermarket in Mid Michigan, in September, 1991. ....................... 175
Table 5.2. Number and description of Italian feed samples. ........................... 176

Table 5. 3. Number and description of fresh corn sample harvested from
five counties in Michigan in Summer, 1994' .................................. 177

 

 

Figure 1.1. ‘

Figure 1.2

Figure 1 3

Figure 14

Figure 1_

Figure 1 5

“WE 1.7. l

I
5

 

LIST OF FIGURES

Figure 1.1. Factors affecting occurrence of mycotoxins in the human food
Chain (Pestka and Casale, 1990) ...................................................... 6

Figure 1.2. Structures of known fumonisins and of Alteman’a altemata f. sp.
chopersici (AAL) toxin, showing structural similarities with
Sphingosine (Norred, 1993) ............................................................. 11

Figure 1.3. Structure and numbering system of some naturally identified
trichothecene mycotoxins (modified from Ueno, 1983). ................... 18

Figure 1.4. Classification of trichothecene mycotoxins based on their
Chemical structure (modified from Ueno, 1980). .............................. 19

Figure 1.5. Preparation of immunogen for (left) fumonisins and (right)
deoxynivalenol (DON) (Pestka et al., 1995) .................................... 43

Figure 1.6. Process of antibody production in vivo (left) and using gene
technology (right). Step 1: rearrangement or assembly of germ
line V-genes; step 2: surface displaying of antibody; step 3:
antigen—driven or affinity selection; step 4: affinity maturation; step
5: production of soluble antibody or antibody fragment (Adapted
from Vlfinter et al., 1994) ................................................................... 48

Figure 1.7. Competitive ELISA for mycotoxins (adapted from Pestka, 1988).
In direct ELISA, enzyme-labeled mycotoxins (ENZ) are
simultaneously incubated with free mycotoxins (D)over solid-
phase bound antibodies (AB). Concentration of free mycotoxins
is inversely related to bound enzyme-labeled mycotoxins and
can be measured quantitatively using spectrophotometer after
color development by the addition of enzyme substrate. In
indirect competitive ELISA, mycotoxin specific antibodies (AB)
compete with free mycotoxins (CI) to solid-phase mycotoxin-
carrier protein (CP) conjugate. Second antibodies (ab-ENZ)
which have been labeled with enzyme are then added to
determine total antibody bound. Concentration of free toxin is
inversely related to bound enzyme-labeled antibodies. ................... 51

Figure 1.8. ELISAGRAM procedure for mycotoxins (Pestka, 1991). ................. 52
xi

 

 

Figure Zl

Figure 2 2

 

Figure 2 3

Figure 274

Figure 2

Figure

Figure 2.1. ELISA titration of rabbit polyclonal FBI antibodies. Sera were
obtained two weeks after the second injection with FBt-KLH
immunogen. Each data point represents the average value of
duplicate measurements. Filled symbols indicate indirect ELISA.
Open symbols are direct ELISA. R1, R2 and R3 refer to rabbit
number. The letter i indicates immunized and p indicates
preimmun ........................................................................................ 99

Figure 2.2. Competitive inhibition ELISA for FB1 using different rabbit
polyclonal antibodies. Sera were obtained two weeks after the
second injection with FBt-KLH immunogen. Each data point
represents the average value of duplicate measurements. Filled
symbols indicate CI-ELISA. Open symbols are CD-ELISA. R1,

R2 and R3 refer to rabbit number. .................................................. 100

Figure 2.3. Competitive inhibition ELISA for F81 using mouse sera. Sera
were obtained ten days after the third subcutaneous (SC)
injection with FBt-KLH immunogen. Each data point represents
the average value of duplicate measurements. Filled symbols
indicate CI-ELISA. Open symbols are CD-ELISA. Subscript 1.4
refers to mouse number. ............................................................... 101

Figure 2.4. Competitive inhibition ELISA for FB1 using mouse sera. Sera
were obtained ten days after the third intraperitoneal (IP)
injection with FBI-KLH immunogen. Each data point represents
the average value of duplicate measurements. Filled symbols
indicate CI-ELISA. Open symbols are CD-ELISA. Subscript 1-4
refers to mouse number. ............................................................... 102

Figure 2.5. Competitive indirect ELISA curves for FB1 using hybridoma
supematants. Supematants were obtained from hybridoma cells
producing FBI antibodies before cloning. Each data point
represents the mean value of duplicate measurements. ............... 103

Figure 2.6. ELISA titration of monoclonal F81 antibodies prepared from
ascites fluid. Each data point represents the mean value of
duplicate measurements. Filled symbols indicate Cl-ELISA.
Open symbols are CD-ELISA. 0105 clone had no titer when
determined with CD-ELISA. .......................................................... 104

Figure 2.7. Competitive inhibition ELISA for F81 using monoclonal
antibodies prepared from ascitic fluid. Data points represent the
mean value of duplicate measurements. Filled symbols were CI-
ELISA. Open symbols were CD-ELISA. Q1CS clone had no
titer when determined with CD—ELISA. .......................................... 105

xii

Figure 2':

 

 

 

I
Figure 2 E

 

Figure 2.1 .'

FIQUTe 2

Figure 2.8. Competitive direct ELISA curves for FB1 using FBt sheep anti
sera (Neogen Corp., Lansing, MI). F8, was dissolved in
extractant [10% methanol (vol/vol.) in distilled water], fresh
corn extract numbers 37 and 56 at a dilution of 1:35). Each data
point represents the mean value of triplicate measurements ......... 107

Figure 2.9. Competitive direct ELISA curves for F81 using F 8, sheep anti
sera (Neogen Corp., Lansing, MI). FBI was dissolved in
extractant [10% methanol (vol/vol.) in distilled water], fresh
corn extract numbers 37 and 56 at a dilution of 1:200). Each
data point represents the mean value of triplicate
measurements. ............................................................................. 108

Figure 2.10. Comparison of competitive inhibition ELISA for F81 mouse-
monoclonal antibodies, rabbit-and sheep-polyclonal antibodies,
and Veratox". Monoclonal antibodies (N205 MAB and 205
MAB) were prepared from ascitic fluids. N205 MAB was
generated in the current study using F BLKLH immunogen, while
205 was produced by Azcona-Olivera (1992a) using F81-
Cholera toxin immunogen. R3 refers to rabbit number 3. Sheep
antiserum was from a single lot supplied by Neogen Corp.
(Lansing, MI). Veratox’ is an ELISA kit for FB1 based on CD-
ELISA and is produced and supplied by Neogen Corp.
(Lansing, MI). Monoclonal antibodies were determined with Cl-
ELISA, but polyclonal antibodies were measured with CD-
ELISA. Each data point represents the mean t standard errors
of the mean (n = 4, two duplicate measurements).
Absorbencies at 0 nglml F81 ranged from 0.675 to 1.250 ............. 109

Figure 2.11. Competitive direct ELISA standard curves for F3. , F82 and
F83 , using F81 sheep antisera. Sheep antiserum was from
Single lot supplied by Neogen Corp. (Lansing, MI). Each data
point represents the average value of triplicate determination ....... 111

Figure 2.12. A typical “broad range “ standard curve used for HPLC
analyses of Fusarium corn cultures. Each data point represents
the average value of two determinations ....................................... 113

Figure 2.13. A typical “low range “standard curve used for HPLC analyses
of corn and corn products. Each data point represents the
average value of two determinations ............................................. 114

xiii

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Figure 3.1. Indirect ELISA titration of mouse antibodies obtained ten days
after the third injection with BSA-HS-DON conjugate. The first
injection was given at a dose of 50 and 100 uglmouse. The
second and third injections were given at a dose of 25 and 50
jug/mouse. Each data point represents the mean :t standard
error of the mean (n = 6, duplicate measurements from three
mice). lP indicates peritoneal injection. SC indicate
subcutaneous injection. 50 and 100 indicate dose (ug) of the
first injection. ................................................................................ 144

Figure 3.2. Indirect ELISA titration of mouse antibodies obtained ten days
after the second (SI) and third immunizations (T I) with BSA-HS-
DON conjugate. The first immunization was given via
intrasplenic deposition at a dose of 20 uglmouse. The second
and third immunization were given via intraperitoneal (IP) and
subcutaneous (SC) injections at a dose of 10 and 50 ug,
respectively. Each data point represents the mean :I: standard
error of the mean (n = 8, duplicate measurements from four
mice). . .......................................................................................... 145

Figure 3.3. Direct ELISA titration of rabbit antisera. Hunter's adjuvant
indicates that rabbits were given a two Sites intramuscular
injection with an emulsion of Hunter Titer Max and BSA-HS-
DON conjugate at a dose of 100 pg per rabbit for both the first
and second injections. Freund’s adjuvant indicates that rabbits
were given a ten site subcutaneous injection with an emulsion
of Freund’s adjuvant and BSA-HS-DON conjugate at a dose of
500 ug and 250 pg per animal for the first and second
immunization, respectively. Antisera were obtained four weeks
after the second injection with BSA-HS-DON conjugate. Each
data point represents the mean 1 standard error of the mean (n
= 6, duplicate measurements from three rabbits). ......................... 146

Figure 3.4. Types of protein-deoxynivalenol conjugation. ................................ 147

Figure 4.1. Competitive indirect ELISA for “trichothecene load” using yeast
ribosomes for coating microtiter plates. Trichothecene
ribosomal binding site specific antibodies (AB) compete with
trichothecenes (T) for the trichothecene binding Site (TBS).
Second antibodies (SAB) which have been labeled with enzyme
(ENZ) are then added to determined total bound antibody (AB).
“Trichothecene load” is inversely related to the bound enzyme-
Iabeled antibodies and can be measured quantitatively using
spectrophotometer after color development by the addition of
enzyme substrate. ........................................................................ 168

xiv

Flt

 

 

Figure 4.2. Inhibition of T-2 toxin to the association of 3H-T-2 toxin to yeast
ribosomes. Each data point represents the mean value of
triplicate measurements. One hundred ul of yeast ribosomes
(0.4 mglml standard buffer) were reacted with 10 pl of 3H-T-z
toxin (0.2 uCiIml) in the presence of 10 ul of serially diluted (0
to 1000 ng) non radiolabeled T-2 toxin and 60 ul of precooled
alcohol. As a control the same amount of radiolabeled toxin
was mixed with the standard buffer without ribosomes. After
ribosome-bound 3H-T-2 toxin was separated by centrifugation,
radioactivity (DPM) of 100 pl of the supernatant was measured
by liquid scintillation counting. DPM values of control solution and
0 nglml supernatant were 16,026 and 8,577, respectively.
Percent inhibition = [(DPM value of certain ng T-2 toxin lml
supernatant - DPM value of 0 nglml supematant)l(DPM value of
control solution - DPM value of 0 nglml supematant)] x 100%. ......... 169

Figure 4.3. Typical indirect ELISA titration of mouse antisera. The mouse
antisera were obtained ten days after the third subcutaneous or
intraperitoneal injection with song of 608 or 808 yeast
ribosomal subunit emulsified with F reund’s adjuvant. Each data
point represents the mean :I: standard error of the mean (n=6,
duplicate measurements of three mice). IP, and SC indicate
intraperitoneal and subcutaneous injections, respectively. 803
and 608 indicates 808 and 608 yeast ribosomal subunits,
respectively. ................................................................................. 170

Figure 4.4. Typical indirect ELISA titration of mouse antisera. The mouse
antisera were obtained ten days after the third subcutaneous
injection with song of 808 yeast ribosomal subunit mixed with
different adjuvants. Each data points represents the mean a:
standard error of the mean (n=4, duplicate measurements of
two mice). 808 indicates 80$ yeast ribosomal subunit. FA, CT,
and T-2 indicate Freund’s adjuvant, Cholera toxin, and T-2
toxin, respectively. .......... . ............................................................. 171

 

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INTRODUCTION

Mycotoxins are a group of secondary metabolites produced by toxigenic
strains of fungi. One fungal genus that contains Species capable of mycotoxin
production is Fusarium. Among the Fusarium mycotoxins that are toxic to both
human and animals and commonly found in grain and grain products are
fumonisins and trichothecenes. The presence of these toxins in agricultural
commodities are primarily dictated by environmental and biologic factors. Since
controlling both environmental and biological factors is almost impossible,
prevention of mycotoxin entries into human foods and animal feeds is commonly
accomplished via detection of the toxins in agricultural commodities. Mycotoxin
detection can be carried out using either conventional methods such as thin layer
Chromatography (TLC), gas chromatography (GC), high performance liquid
Chromatography (HPLC), and mass spectroscopy (MS) or immunoassay
techniques such as enzyme-linked immunosorbent assay (ELISA). ELISAS are
commonly preferred to conventional methods because of simplicity, rapidity, and
applicability both in fields and laboratories.

ELISAS for fumonisin 81 (FB1) have been developed in our laboratory.
However, when these ELISA systems are used to detect F31 in fusarium corn
cultures, corn and corn products, they provide much higher F81 estimates than

HPLC methods. One possible explanation for these observations is that F 81

 

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antibodies used in these ELISAS are not completely specific to fumonisins and,

therefore, may have reacted with other compounds found in Fusarium cultures,
and contaminated corn. To overcome this problem, it is desirable to produce
antibodies which have higher affinity and specificity to the toxins.

Our laboratory has also developed an ELISA for deoxynivalenol
trichothecene. When this ELISA was used to detect deoxynivalenol (DON) in
com-based foods, it had a relatively high detection limit (1000 ppb). A lower
detection limit of an ELISA can be generated when DON antibodies used in the
ELISA have a higher affinity and specificity to DON. Therefore, production of
higher affinity and specificity antibodies against DON is desirable.

Trichothecenes are a potent inhibitor of eukaryotic protein synthesis
because they bind to a common site on the 603 ribosomal subunit. These toxins
compete with each others for the ribosomal binding site in proportion to their
toxicity. Production of antibodies Specific to the binding site would enable
investigators to develop an ELISA that can assess total ”trichothecene load” in
agricultural commodities and, thus enhance food safety.

The research in this dissertation was undertaken to address all above
rationales and this dissertation was divided into four parts: Part I (Review of
Fusarium Mycotoxin Immunoassay); Part II (Development and Application of an
ELISA for Fumonisin B1 in Fungal Cultures, Corn and Corn Products); Part III
(Production of Deoxynivalenol Antibodies); and Part IV (Production of Antibodies
against Trichothecene Yeast Ribosomal Binding Site). Part I reviews Fusarium

mycotoxins especially fumonisins and trichothecenes including their chemical

structures
in detail ’5
develop"
com Cult;

antibodre

 

respect“

 

 

 

3
structures, toxicity, natural occurrence, and detection methods. Part II describes

in detail: the production of F B1 antibodies, the use of the antibodies for ELISA
development, and the application of the ELISA for detection of F81 in Fusarium
corn cultures, corn and com product. Part III and IV explain efforts to produce
antibodies against deoxynivalenol and trichothecene yeast ribosomal binding Site,

respectively.

PART I.

REVIEW OF FUSARIUM-MYCOTOXIN IMMUNOASSAY

 

 

Introductir
My:
part oi se
contamin;
and Mos:
products
Ne
l0 comm:
environmt
usual/y 0c
favor certa.i
1983'. Shob
environment;
The human foc
MIcolo
lllllllan and fa
lldude: (1)
“II/30mm, (2

litterial or _

 

FUSARIUM MYCOTOXINS

Introduction

Mycotoxins are a very diverse group of toxic compounds produced as a
part of secondary metabolism in a wide variety of filamentous fungi which often
contaminate agricultural commodities prior to harvest and during storage (Smith
and Moss, 1985). Peanut, corn, feeds, wheat and cereals are five agricultural
products which most often have mycotoxin problems (Hesseltine, 1986).

Natural occurrence of mycotoxin in foods and feeds varies from commodity
to mmmodity, year to year and region to region and are strongly dictated by
environmental factors (CAST, 1989). Trichothecene mycotoxin contamination
usually occurs during cold and wet season, or in extreme drought years which
favor certain fungus infections and toxin production (Vesonder et al., 1978; Ueno,
1983; Shotwell et al., 1985; Tanaka et al., 1988a; CAST, 1989). Effects of
environmental conditions and genetic factors on the occurrence of mycotoxins in
the human food Chain are illustrated in Figure 1.1.

Mycotoxin-contaminated products can produce adverse effects on both
human and farm animals when the products are consumed. The effects may
include: (1) acute toxicity and death following high level exposures of a
mycotoxin, (2) lower growth rate, impaired immunity, greater susceptibility to

bacterial or parasitic infection, (3) decreased milk and egg production,

 

 

. HI & #\
era/22%

OOH!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

Biological Environmental Harvesting
Factors Factors
Susceptibile Temperature Crop Maturity
Crop + ' Moisture ' Temperature
Compatible. Mechanical Injury Moisture _
Toxigenic Insect/Bird Damage Detection/Diversion
Fungus Fungus
Storage
Temperature
Moisture ‘
Detection/Diversion
Distribution-
Processing
“ Detection/Diversion
Humans < . Animals

Animal Products

Figure 1.1. Factors affecting occurrence of mycotoxin in the human food Chain
(Pestka and Casale, 1990)

 

 

 

(4) reduced I
location eta
1989). Con:
livestock prod
The e
livestock log
heath lCAS‘
human food
Cosmetic A;
established
m leVel:
(CAST~198
diatoms, .
melamine C
bwnmsa
all? (300 p
bleeding cat-ill
AmOng
zearalenOne‘ a
first ”7’98 mic;

especially mm

 

7
(4) reduced reproductive efficiency, or (5) Chronic toxicity such as cancer

formation after prolonged exposure to small quantities of the mycotoxins (CAST.
1989). Consequently, mycotoxins cause economic losses to farmers and
livestock producers (Hesseltine, 1986).

The economic losses (Table 1.1) are derived not only from crop and
livestock losses, but also from regulatory action to protect human and animal
health (CAST, 1989). In the United States, for example, the amount of aflatoxins in
human foods and animal feeds has been regulated. Under the Food, Drug, and
Cosmetic Act, Section 402(a)(1), the US. Food and Drug Administration (FDA) has
established acceptable aflatoxin levels in agricultural commodities by establishing
action levels that allow for the removal of violative lots from interstate commerce
(CAST, 1989). The action levels of human foods are 20 part per billion (ppb) total
aflatoxins, except for milk which has an action level of 0.5 ppb for aflatoxin M (a
metabolite of aflatoxin 81). The action level of aflatoxins for feeds is 20 ppb, except
for cottonseed meal used in feeds (300 ppb), corn used for finishing (feedlot) beef
cattle (300 ppb), com destined for finishing swine (200 ppb), and feeds used for
breeding cattle, breading swine, and mature poultry (100 ppb)(CAST, 1989).

Among the most important mycotoxins are fumonisins, trichothecenes,

zearalenone, aflatoxins, and ochratoxins (Hesseltine, 1986; Pohland, 1993). The
first three mycotoxins are produced by Fusan‘um spp. Fusarium mycotoxins

especially fumonisins and trichothecenes will be reviewed in this chapter.

Bearer

____________.__
Farmers a“:
stool orOC-JC

 

Fooc am ‘e
rartdle’s :1
at prices

Sevemmer

 

 

 

 

 

 

 

8

Table 1.1. Types of economic losses and costs associated with mycotoxin
contamination in foods and feeds (CAST, 1989)

 

Bearer

Economic losses and costs

 

 

Farmers and live
Stock producers

Food and feed
handlers, distributor
and processors

Government

Consumers (human
or animal)

 

- Outright food and feed loss.

- Contaminated crops provide less income and may lead to potential
losses of outlet.

- Reduced productivity of livestock from (1) lower quantity and quality of
animal products, (2) smaller letters, (3) reduced work output, (4) loss of
pregnancy, (5) reduced feed efficiency, (6) impaired resistance to
disease, and (7) loss of vaccination efficacy.

- Less income from products refused, condemned, or sold at discount.

— Increased storage, transport, and packing costs on such products.

- Potential loss of market, trading reputation, and raw material source.

- Increased costs due to litigation (may exceed cost of product),
surveillance, and control.

- Lower foreign exchange earnings from reduced exports.

- Increased cost involved in shipment, sampling, and analyses of exported
goods that are subsequently refused import entry; potential loss of
overseas outlets.

- Increased costs of detoxification or reconditioning abroad.

- Increase costs for food or feed imports; staple food subsidies.

- Increased costs of surveillance and mntrol.

- Increased costs for expenditures on human and animal health facilities
and activities.

- Increased costs involved in training and extension programs.

- Consumption may lead to impaired health and productive capacity.

- Lack of food may lead to undemutrition or higher food prices resulting
from outside purchase of foods or feeds.

- Possible medical and veterinary costs associated with the above
conditions in previous two statements.

- Possible consumer-initiated litigation costs.

 

 

 

 

Furllonisins

Histo
metabtllites
19881. and‘
lound on 0
lila'asas 8'
Sheldon (1!
agent of ‘rr
mold hasb
equine le;
reurotoxioc
matter of r
Slurptoms
tausatrve a
{Benzuidenl
in fumonisrn.
dIStnbUIIOII, p

FBI is ,-
Mon/l/fome (G
agent gi (am a
WWW/”Toni

Ill
93/69/5194) 1,

   

Fumonisins

Historical background. Fumonisins are a group of secondary
metabolites produced by Fusarium moniliforme (Sheldon)(Benzuidenhout et al.,
1988), and Fusarium proliferatum (Ross et al., 1990). These fungi are commonly
found on corn, sorghum, and other grain commodities throughout the world
(Marasas et al., 1984a). Fusarium monilifonne, which was firstly described by
Sheldon (1904) as Fusarium monilifonne Sheldon, was implicated as a causative
agent of “moldy corn poisoning” in animals in the United State (Peter, 1904). This
mold has been associated with poisoning in equine Species that is now known as
equine leukoencephalomalacia (ELEM) (Ross, 1994). This disease is a
neurotoxicosis that is characterized by multifocal liquefactive necrosis in the white
matter of cerebral hemispheres (Marasas et al., 1988b). Although the disease
symptoms have been observed since the previous century (Marasas, 1986), its
causative agent [fumonisin 81 (F B1 )] was not isolated and identified until 1988
(Benzuidenhout et al., 1988; Marasas et al., 1988b). As a consequence, interest
in fumonisins has increased leading to extensive studies on their occurrence,
distribution, production, toxicity, detection, and chemistry (Ross, 1994).

F81 is the most toxic and predominant fumonisin produced by Fusarium
moniliforme (Gelderblom et al., 1988b), and has been confirmed as an etiologic
agent of fatal animal diseases such as ELEM in horses (Kellerman et al., 1990),
porcine pulmonary edema (PPE) in pigs (Harrison et al., 1990), and liver cancer
in rats (Gelderblom et al., 1991). In addition, epidemiological studies indicate that

fumonisins are associated with human esophageal cancer in South Africa

(Marasas et 5
given in Part I

Chem
add and ei‘.
pentahydrox

Chemical st

 

troopers/cl

 

studies. to
were isoia‘
Marasas (
al., 1991:
been rept
whereas t
a methyl I
fresco r.
Madman
The

0th been p,

 

1994) SDI/d SI

10
(Marasas et al., 1988a). A detailed review of fumonisin toxicity and detection is

given in Part II.

Chemistry. Fumonisins are diesters of 14, 15-propane-1,2,3-tricarboxylic

acid and either 2-acetylamino- or 2-amino—12, 16-dimethyl-3, 5, 10, 14, 15-
pentahydroxy-icosane or its C-10 dery analogue (Bezuidenhout et al., 1988).
Chemical structures of these toxins are similar to that of Alteman'a altemata f. Sp.
chopersici (AAL) toxin and sphingosine (Figure 1.2)(Norred, 1993). In early
studies, four fumonisins (FA1, FA2, F81 and FB2 ) (Bezuidenhout et al., 1988)
were isolated and identified by the South African researchers led by Dr. W.F.O.
Marasas (Norred, 1993). Additional chemical structures for F83, FB4 (Cawood et
al., 1991; Plattner et al., 1992) and F01 (Branham and Plattner, 1993) have now
been reported. The A-series fumonisin are acetylated at the amino group
whereas the B-fumonisins have a free amine (Figure 1.2). F01 is F B1 which lost
a methyl group at C-1 position; thus F01 is a diester of 13, 14-propane-1,2,3-
tricarboxylic acid and 1-amino-11, 15-dimethyl-2, 4, 9, 13, 14-pentahydroxy—
nonadecane (Branham and Plattner, 1993).

The biosynthetic pathways through which fungi produce fumonisins have
only been partially elucidated (Norred, 1993). When Fusarium spp. are cultured
on solid (corn) substrates, FB1 is produced predominantly by Fusan'um
moniliforme strains, but some strains of Fusarium proliferatum produce FB2 or
F33 at higher concentrations than F81 (Nelson et al., 1994; Visconti and Doko,

1994). Solid substrate media are not conductive for the study of fumonisin

FULIC

 

 

SPI

“the 1.2

 

11

FUMONISINS (itOOH

 

 

COCH,CHCH,COOI~I
\ R. R; R. I . .
FA. OH OH CH,CO “- “ or,
H OH Waco ”ONFHEMUTI“.OCTI' I
,, NHR,
Pei. OH OH H “04'? CH’ 8‘
e. t. a. H mmcwr
‘ H
FB. .OH H H
FB. I-I - H H
('ZOOH
COOLOIOLCOOII
' ' on OH
AAL TOXIN W
C“. CH CH» 0H NH.
OH
SPHINGOSINE /\/\/\/\/\/\/W :.' "
. ' NH;

FIgure 1.2. Structures of known fumonisins and of Alteman‘a altemata f. sp.
chopen’sici (ALL) toxin, showing structural similarities with spingosine
(Norred, 1993). A

libs
Cultu
the m
Sin/tar
tidings

shines;

581/76, [3

 

12
biosynthesis, and production of fumonisins in liquid media must be performed

(Plattner and Shackelford, 1992; Blackwell et al., 1994). When Plattner and
Shackelford (1992) fed deuterium-labeled methionine to Fusarium monilifonne
cultures, they observed high levels of deuterium incorporation into F81 at the
methyl groups on the C-12 and C-16 position of the fumonisin backbone. Since
the'structure of fumonisins is Similar to that of sphingosine, these investigators
speculated that biosynthesis of a fumonisin backbone is also Similar to that of
sphingosine. Sphingosine is synthesized through condensation of linolyl-
coenzyme A and alanin, whereas the fumonisin backbone is synthesized via
condensation of palmitoyl coenzyme A and serine (Plattner and Shackelford,
1992)

However, the above hypothesis was contested by Blackwell et al. (1994)
who fed 13C-Iabeled acetate to liquid cultures of Fusarium moniliforme to
determine the location of labeled carbon atoms in the radiolabeled fumonisin.
They observed that the methyl group (C-2) of acetate resulted in enrichment of C-
20, 18, 16, 14, 12, 10, 8, 6, and 4 while the carbonyl group (C-1) of acetate
labeled C-19, 17, 15, 13, 11, 9, 7, 5, and 3. When they added methionine to the
cultures, F81 production increases up to 12-fold, with the S-methyl group from
the methionine exclusively enriching positions C-21 and C22. Those resutls were
Similar to the results of Plattner and Shackelford (1992). Based on these
findings, Blackwell et al. (1994) concluded that the backbone of fumonisins was
synthesized by the fungus through condensation of acetyl coenzyme A and

serine, rather than modification of palmitic acid as stated by Plattner and

 

reo

an 1
FBI

non/i

ion it

than t

rarities

11051 I lie

 

13
Shackelford (1992). More work is still required to fully elaborate the biosynthetic

pathway of the fumonisins.

Natural occurrence. Fusarium monilifomIe , the fumonisin-producing
fungus, commonly occurs in the world and it has been isolated from many
countries including Australia, Brazil, Canada, Central America, China, Croatia,
Egypt, German, Hong Kong, India, Indonesia, Israel, Italy, Jamaica, Japan,
Nepal, New Zealand, Peru, Philippines, Poland, Portugal, Romania, South Africa,
Taiwan, the United State, Turkey and Zambia (Bacon and Nelson, 1994; Doko et
al., 1995). This fungus is primarily found as a corn contaminant, but can also be
found on several grain commodities such as sorghum, wheat, rice, and oat and
on other agricultural products such as beans, peanuts, sugar beats, and bananas
(Bacon and Nelson, 1994). Natural occurrence of fumonisins has been
documented in a number of countries (Table 1.2).

Conditions required for optimal production of fumonisins in the field or
storage are only partially known (Bacon and Nelson, 1994). Bars et al. (1994)
reported that a temperature of 20°C, a 32% moisture content of com media, and
an aerated atmosphere (cotton-stoppered flasks) were the optimum condition of
F Bi production by Fusarium monilifomie in laboratory experiments. Fusarium
monilifomie that was isolated from European fresh corn produced FB1 up to 300
ppm when cultured on solid corn media at the optimum conditions for 12 days
(Bars et al., 1994). Other Fusarium moniliforme isolates produce higher
quantities (6400 ppm) of FB1 when cultured on the same media (Nelson et al.,

1991). More higher F 81 (17,000 ppm) was produced when Fusarium moniliforme

 

 

f05’t’t

lnterr
0f tor.

ttreat

 

14
MRC 826 was cultured on corn media at 20°C for 13 weeks (Alberts, et al.,

1990)

Trichothecenes

Historical background. Trichothecenes are a group of structurally similar
sesquiterpenoid metabolites produced mainly by Fusarium spp. (Bamburg and
strong, 1971; Bamburg, 1983; Ueno, 1983; Betina, 1989). The first known
member of this group, trichothecin, was originally dismvered as an antifungal
antibiotic in 1948 (Freeman and Morrison, 1948; 1949). Since that time over
eighty trichothecenes have been isolated and characterized (Betina, 1989).

Interest in trichothecenes has increased over the years since their first
discovery. The former Soviet Union was the first country to conduct an extensive
research on these toxins in attempts to identify and elucidate the causative
agents of mycotoxicoses in the 1930’s following outbreaks of alimentary toxic
aleukia (ATA) and stachybotriotoxicoses (Ueno, 1980; Bamburg, 1983). Japan
followed shortly thereafter because this country also suffered from a
trichothecene-mediated disease known as red-mold (akakabi in Japan) disease.
Interest in mycotoxins in the Western worId did not emerge until the appearance
of “turkey x disease”, which killed thousands of turkeys in the United Kingdom in
the early 19605 (CAST, 1989). The etiologic agent of the “turkey x disease” was
feed contaminated by Aspergillus flavus which produced aflatoxins (Spensley,
r963)

 

 

 

 

 

 

 

 

 

 

 

‘1:
IV

Table 1.2. Natural occurrence of fumonisins in cereals and cereal products

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CROP GRAIN MEAN IN POSITIVES, PPM
COUNTRY YEAR PRODUCTS (POSITIVES/SAMPLES) REFERENCE
F B, FB2
_A_rgentina 1991 com 2.88 (17/17) 1.14 (17/17) Sydenham et al., 1993
Canada 1990 cornmeal 0.05(1/2) nd (0/2) Sydenham et al., 1991
China:
Linxian 1989 corn 0.87 (13/27) 0.45 (3/27) Yoshizawa et al., 1994
Cixian 1991 moldy corn 93.13 (4/4) - Chu and Li, 1994
Shaggqui 1989 corn 0.89 (5/20) 0.33 (1/20) Yoshizawa et al., 1994
Shangqui 1991 moldy corn 5500(5/5) - Chu and Li, 1994
Croatia 1992 corn 0.02 (11/19) 0.01 (4/19) Doko et al., 1995
mi 1990 cornmeal 2.38 (2/2) 0.60 (ZIZ) Sydenham et al., 1991
Italy 1989- corn 0.38 (26/26) 0.14 (13l26) Doko et al., 1995
1991
1991 corn feeds 1.14 (23/25) 0.30 (13/25) Minervini et al., 1992
Peru 1990 cornmeal 0.66 (1/2) 0.14 (1/2) Sydenham etal.,1991
Poland 1992 com 0.02 (2/7) 0.01 (1/7) Doko et al., 1995
Portugal 1992 corn 1.03 (9/9) 1.21 (8/9) Doko et al., 1995
Romania 1992 corn 0.01 (3/6) 0.01 (1/6) Doko et al., 1995
South Africa 1985 com 1.60 (12/12) 0.50 (10l12) Sydenham et al., 1990
1985 moldy corn 29.3(12/12) 7.55(12/12) - -
1989 corn 1.53 (5/6) 0.42 (5/6) Rheeder et al., 1992
1989 moldy corn 53.74(6/6) 1368(6/6) - -
1989 export corn 0.29 (28/68) 0. 13 (1 0/68) Rheeder et al., 1994
1990 cornmeal 0.14 (46/52) 0.08 (11/52) Sydenham et al., 1991
1990 corn grits 0.13 (10/18) 0.09 (4/18) -"-
1990 comflakes nd (013) nd (0/3) - -
Switzerland 1986- corn 2.88 (7/7) 0.24 (7/7) Stack and Eppley, 1992
1991
USA 1989 corn 0.64 (7/7) 0.18 (6/7) Sydenham et al., 1991
1990 cam 55.40 (616) 15.08 (7/7) Stack and Eppley, 1992
scree_niflgs
1990 com foods 0.43 (25/36) 0.14 (18/36) - -
1990 corn food' 0.41 (4/4) 0.15 (3/4) Sydenham et al., 1991
1990 cornmeal 1.05 (15/16) 0.30 (13/16) - -
1990 corn grits 0.60 (10/10) 0.38 (5/10) -"-
1990 comflakes nd (0/2) nd (0/2) - -
1991 cornmeal 0.09 (2/7) nd (0/7) Pittet et al., 1992
1991 corn grits 0.26 (34/55) 0.10 (13I55) - —
1991 comflakes 0.06 (1/12) nd (0/12) -”—
1991 sweet corn 0.07 (1/7) nd (0!?) -"-
1991 poultry feed 0.24 (6/22) 0.09 (2/22) - -
1991 corn foods 0.55 (11/13) 0.13 (10/13) Stack and Eppley, 1992
Zambia 1992 Corn 0.18 (20/20) 0.05 (15/20) Doko et al., 1995

 

nd indicate not detected.

- indicate not analyzed.

-”- same as above

 

tin:

 

argued
IIEW an
contamin
'l’9llow ra

The

 

alienating
natural occ
WI. Bas
Weed tr
toll In hum a r

16
Interest in trichothecenes arose world wide in September, 1981 after the

United States indirectly accused the Soviet Union, \ertnam and Laos
governments of employing these mycotoxins as chemical warfare agents
(Bamburg, 1983; Watson et al., 1984). The accusation was based on several
findings. Firstly, analyses of blood, urine, and internal tissues of “yellow rain”
attack victims revealed extremely high levels of trichothecenes (Watson et al.,
1984). Secondly, a high level (up to 20 times higher than any recorded natural
outbreak) of trichothecene mycotoxins was also found in a Single leaf and stem
sample taken from a region of Kampuchea where “yellow rain” was reported
(Seagrave, 1981). Thirdly, normal background levels of these toxins were
essentially undetectable and natural occurrence of trichothecenes in Southeast
Asia was absent (Bamburg, 1983).

However, a group of researchers disagreed with the above findings. They
argued that “yellow rain” was merely naturally occurring bee feces where fungus
grew and produced trichothecenes. In addition, consumption of trichothecene-
contaminated foods might have yielded similar symptoms to the symptoms of
“yellow rain” attack victims (Schiefer, 1988).

The controversy resulted in intensive research activities directing towards
elucidating many aspects of trichothecene mycotoxins including their detection,
natural occurrence, toxicological, biological and biochemical action (Bamburg,
1983). Based on this and other research over the last five decades, it is now
recognized that trichothecene mycotoxins are etiologic agents of mycotoxicoses

both in human and animals (Ueno, 1980; 1983; Bamburg, 1983; CAST, 1989).

that
13 I
Bet

natu

 

toxm

posit

numb

 

Betina

dassifr
1.4). t
toxin ar
deoxynn
such asr
beMeenc
The
generally 5
Cllloroform’
l97l.’ Ueno,
aIcohol den
homologue

leadlly 83%“

 

17
Chemistry. Trichothecenes all have tetracyclic, sesquiterpenoid structure

that includes a six-member oxygen containing ring, an epoxide group in the 12,
13 position, and an olefinic bond in the 9, 10 position (Ueno, 1980; 1983;
Bamburg, 1983; Betina, 1989). Structure and numbering system of some
naturally identified trichothecene myCotoxins are illustrated in Figure 1.3. These
toxins possess oxygen-containing substitutes at one or more C-3, 4, 7, 8, and 15
positions. The substitutes may be hydroxyl, esterified hydroxyl, keto (carbon
number 8 only), or epoxide (carbons number 7, 8 only) groups (Ueno, 1983;
Betina, 1989).

Based on trichothecene structural characteristics, Ueno (1980; 1983)
classified trichothecenes into 4 different groups (group A, B, C and D; Figure
1.4). Group A trichothecenes have hydroxyl or acetoxy substitutes, such as T-2
toxin and trichodermin. Group B trichothecenes are 8-keto derivatives such as
deoxynivalenol and nivalenol. Group C trichothecenes are 7,8 epoxide derivatives
such as crotocin. Group D includes trichothecenes containing a macrocyclic ring
between carbon number 4 and 5 such as verrucarin A and roridin A.(Ueno, 1980).

Trichothecenes are colorless, crystalline, optically active solids, and are
generally soluble in non-polar solvents such as acetone, ethylacetate, and
chloroform, but less soluble in polar solvents (e.g. water) (Bamburg and Strong,
1971; Ueno, 1980). Trichothecenes are more stable in solid condition. Their
alcohol derivatives have a higher solubility in water than their esterified
homologue (Betina, 1989). Under a mild alkaline solution the ester groups are

readily saponified and result in less acylated derivatives or parent alcohol forms

 

 

Ilia
Sci
Deo
(v01
Nive
11155

Figure

 

 

18

 

 

 

 

j-"

 

Ezicnognecene R1 R2 R3 R4 R5

T-Z toxin on OAC OAC H ooccu,ca (CH3) ,
arr-2 toxin on on OAC H ooccnzcn (C8,) ,
T-Z tetraol OH OH OH H OH
Diacetoxyscirpenol OH OAC OAC H H
Scirpenetriol OH OH OH H H
Deoxynivalenol .
(vomitoxin) OH H OH OH O
Nivalenol OH OH OH' OH O
Fusarenon-x OH OAC OH OH 0

Figure 1.3. Structure and numbering system of some naturally identified
trichothecene mycotoxins (modified from Ueno, 1983).

 

 

Figure I.

19

 

     

0
R1

11’
RI

Group D

Figure 1.4. Classifiwsion of trichothecene mycotoxins based on their chemml
structure (modified from Ueno,1980).

20
(Ueno, 1980). The double bond at carbon 9, 10 can be catalytically reduced to

a dihydro derivative, and the corresponding alcohol groups are then easily
oxidized to ketone or aldehyde functional groups.

Natural occurrence. Only a few members of the trichothecenes, such as
deoxynivalenol (DON), nivalenol (NIV),T-2 toxin (T-2), HT-2 toxin, and
diacetoxyscirpenol (DAS) are detected as natural contaminants in cereal grains
although over 80 members of these toxins have been characterized in the
laboratory (Betina, 1989).

Production of trichothecenes in cereal grains is mainly dictated by
environmental conditions (Vesonder et al., 1978; Ueno, 1983; Cote et al., 1984).
Wet and cool seasons favor Fusarium infection and trichothecene production in
the field (Tuite et al.,1974; Shotwell et al., 1977, 1983; Ueno, 1983; CAST, 1989).
Tuite et al. (1974) reported that during the unusually cool and wet summer of
1972, corn produced in the United States was heavily infected with Fusarium
gram/nearum, and caused outbreaks of feed refusal and emesis in swine. DON
was believed to be an etiologic agent for these outbreaks. Shotwell et al. (1977,
1983) observed Similar effects of weather on Fusarium contamination in wheat,
grown in VIrginia from 1975 to 1980. The weather was unusually cold and rainy
during the 1975 growing season, but during the following 5 years (1976 to 1980)
such weather did not occur. These investigators found zearalenone in 19 of 42
samples from wheat grains harvested in the cool and wet growing season;

however, they did not find the toxin during the 1976-1980 harvest period. Later,

DON was also detected in the zearalenone-positive samples (Shotwell and

 

 

 

()

21
Hesseltine, 1983). “Red mold disease” in Japan that is caused by Fusarium

gramineanrm and Fusarium nivale (Ueno, 1983), also occurred after a long rainy
season in 1963 and in 1970. Similar weather conditions resulted in “scabby
wheat” (soft and shriveled wheat and often with a pink discoloration) in the United
States and Canada (CAST, 1989). This “scabby wheat” was caused by Fusarium

gram/nearum which produced DON (Shotwell et al., 1985; Tanaka et al., 1988a;
Fernandez et al., 1994).

The natural occurrence of DON as well as NIV in cereal and cereal

products has been surveyed by several investigators in several countries and the

results are summarized in Table 1.3.

Human and animal mycotoxicoses. Outbreaks of human and animal
diseases associated with consumption of trichothecene-contaminated foods and
feeds occurs in many countries usually after a cold and wet harvest season
(Bamburg, 1983). Although such outbreaks have been reported to take place
Since the late 19'h century (Bamburg, 1983; Ueno, 1983), elucidation of
trichothecenes as an etiological agent in mycotoxicoses was not reported until
1972. At that time, Hsu et al. (1972) demonstrated that 2 ppm of T-2 toxin, a
trichothecene produced by Fusarium tn'cinctum, was detected in moldy corn
associated with illness and death of lactating cows. Since that time other
trichothecenes such as diacetoxyscirpenol, nivalenol, fusarenon-X,

deoxynivalenol and HT-2 toxin have also been found naturally in feedstuffs and in

cereal grains (Betina, 1989).-

22

Table 1.3. Natural occurrence of trichothecene deoxynivalenol (DON) and

nivalenol (NIV) in cereals and cereal products

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CROP GRAIN MEAN IN POSITIVES, PPM
COUNTRY YEAR PRODUCTS (POSITIVES/SAMPLES) REFERENCE
DON NIV
Argentina 1983 wheat 0.02(3l20) nd (0/20) Tanaka et al., 1988b
1983 barley 024(18/20) 0.03(15/20) - -
1983 corn 011(2/20) nd (0/20) - -
Austria 1983 wheat 0.36(3/4) 0.03(3/4) Tanaka et al., 1988b
Bulgaria 1983 wheat 0.21 (1/2) 0.03(1/2) Tanaka et al., 1988b
Canada 1980 wheat 030(72/72) - Scott et al., 1981
1980- wheat 1 .26(9/10) 0.02(4/10) Tanaka et al., 1988a
1984
1984 corn 0.96(1I1) 0.01(1l1) - -
1982 rye 0.20(1/1) 0.01(1l1) Tanaka et al., 1988b
China 1984 wheat 1.71 (1 [5) 6.64(1/5) Ueno et al., 1986
1985 wheat flour 0.19(7/7) nd (0”) - -
1984 wheat 4.28(4/4) 016(3/4) Tanaka et al., 1988b
England 1984 wheat 0.03(20/31) 0.10(17/31) Tanaka et al., 1988b
Finland 1987- cereal 0.13 - Hietanlemi and
1978 (246/268) Kumpulainen, 1991
France 1984 wheat 0.09(1/2) 0.04(2/2) Tanaka et al., 1988b
Germany 1984 wheat 0.71(2/8) 0.27(0/8) Tanaka et al., 1988b
1984 barley 0.19(2/13) 0.04(1/13) - -
1984 cat 0.14(4/10) 1.45(1/10) -"-
1984 sonan nd (0/1) nd (0/1) -'-
1984 rye 0.41(4/22) 0.01(4/22) - -
1987 barley 0..40(43/44) 0.01(5/44) Muller and
Schwardorf, 1993
1984 rye flour 0.17(1/1) 0.01(1l1) - -
Greece 1984 wheat 0.01(1l1) 0.01(1l1) Tanaka et al., 1988b
Hungary 1984 wheat 0.67(2/2) 0.01(1l1) - -
India 1989 sorghum nd (0/150) - Ramakrisna et al.,
1 990
1989 cam nd (0/102) - -”--
1989 wheat 0.31 (1/58) - -"-
1989 whole - -”-
wheat flour 438(1 1/37)
1989 feed sample nig01102) - - -
Italy 1984 wheat 012(1/12) nd (0/12) Tanaka et al., 1988b
1984 barley 0.19(2/5) 0.02(1/5) - -
1984 corn 0.40(2l3) nd (0/3) -”-
1984 cat nd (015) nd (0/5) -"-

 

nd indicates not detected.

-"- indicates the same as above.

- indicates not analyzed.

 

Table 1.3. Continued

23

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CROP GRAIN MEAN IN POSITIVES, PPM
DON NIV

Japan 1983 wheat 0.02(4/6) 0.39(6/6) Tanaka et al., 1985.
1983 barley 0.25(5/5) 0.71 (5/5) - -

Korea 1983 wheat 0.01(2/10) 0.14(9/10) Lee et al., 1985
1983 rye 0.01(5/5) 0.08(5/5) Lee et al., 1985
1984 wheat 0.02(5/9) 053(9/9) Lee et al., 1986
1983 barley 012(26/28L 055(28/28) Lee et al., 1985
1984 barley 012(31/31) 0.50(31/31) Lee et al., 1986
1989 barley 0.26(2/11) 0.30(3/11) Park et al. 1991
1989 rice nd (0/8) nd (0/8) - -
1989 corn 0.62(1/3) 035(1/3) —'-
1989 millet 0.34 (1/6) 023(1/6) - —
1990 barley 0.19(24/27) 1.11(27I27) Park et al., 1992
1993 barley 0.17(?/39) 1.01 (7/39 Kim et al., 1993
1993 corn 0.31(?/46) - - -

Nepal 1984 wheat 0.06(1/1 0) 0.07(5/10) Tanaka et al., 1988b
1984 barley nd (0/4) 0.02(1/4) - -
1984 cat I'Id (0/7) 0.02(4/7) -"-
1984 rice nd (0/9) 0.02(2/9) -”-
1983 rye nd (0/2) nd 0/2) -"-
1984 com 0.54(3/9) 0.89(6/9) - -

Netherlands 1984 cereals 0.22(26/29) - Tanaka et al., 1990
1988- feeds 0.63(19/95) - Veldman et al., 1992
1989

Poland 1984 wheat 010(13/48) 0.05(37/48) Tanaka et al., 1988b
1984 barley 0.39(1/6) 0.08(3/6) Tanaka et al., 1988b

Pmm 1984 wheat nd (0/4) nd (0/4) Tanaka et al., 1988b

Scotland 1984 wheat 0.03(1/20 nd (0/2) Tanaka et al., 1986
1984 barley 0.04(5/8) 0.39(3@ - -

S. Africa 1987 wheat 0.87(3/3) - Sydenham et al.,

1989

Sweden 1984 wheat nd (0/1) nd (0/1) Tanaka et al., 1988b

Taiwan 1984 wheat 056(9/12) 0.07(6/12) Ueno et al., 1986
1985 barley 0.08(4/4) 0.63(4/4) - -
1985 wheat 0.25(3/10) 0.02(4/10) - -

USA 1977 corn 230(24/52) - Vesonder et al., 1978
1981 cam 3.1(274/342) - Cote et al., 1984
1982 wheat 1.7891/33) - Hagler et al., 1984
1982 wheat 1 .17(45/161) - Shotwell et al., 1985

 

 

 

 

 

9le

Table 1.3. Continued

24

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CROP GRAIN MEAN IN POSITIVES, PPM
COUNTRY YEAR PRODUCTS (POSITIVESISAMPLES) REFERENCE
DON NIV
1984 wheat 0.59(75/123) - Wood and Carter,
' 1989
1984 corn 0.39(61/92) - -”-
1984 dairy grain 0.10(60/101) - Whitlow et al., 1986
1 985 wheat 0.49( 57/ 1 24) - Wood and
Carter, 1 989
1985 corn 047(32/106) - - -
1989 human food 4.00(46/92) - Abouzied et al., 1991
1991 wheat 1.57(17/81) - Fernandez et al.,
1 994
1990 corn 240(13/99) - Price et al., 1993
1990 winter 2.4(201/207) - - -
wheat

1990 sping wheat 0.9(1 201206) - - -

USSR 1984 cat nd (0/2) nd (0/2) Ueno et al., 1986
1984 spice nd (0/3) nd (0/3) Tanaka et al., 1988b
1986 wheat 0.59(6/140 - Tutellyan et al., 1990
1987 wheat 0.26(14/90) - - -
1988 wheat 1 .13(14162) - - -

Yemen 1983 sorghum nd (0/6L 0.09(1/6) Tanaka et al., 1988b
1984 wheat 0.00(1/7) nd (0/7) - -
1984 barley 0.02(2/3) 0.01(2/3) -”-
1984 com 0.01(1/12) nd (0/12) -"-
1984 sorghum nd (0/5) nd (05) -”-
1984 soybean nd (0/2) ndJOQ) -"-
1984 sesame nd (0/7) nd (0/7) -"-

 

nd' indicates not detected.

-"- indicates the same as above.

- indicates not analyzed.

 

25
Severe mycotoxicosis induced by trichothecene mycotoxins include

alimentary toxic aleukia (ATA) in Russia (Joffe, 1962; 1965), red-mold disease in
Japan (Bamburg et al., 1969), moldy corn diseases in The United States
(Bamburg et al., 1969; Hsu et al., 1972), staggering grain toxicosis in Eastern
Siberia (Bamburg, 1983), dendrochiotoxicosis in Russia (Bamburg, 1983),
vomiting and feed refusal in the United States and other countries (Vesonder et
al., 1973), been hull poisoning in Japan (Ueno, 1980), and gastrointestinal
disorders in Kasmir Valley, India (Bhat et al., 1989). Symptoms, victims, and

etiologic agents of these mycotoxicosis are summerized in Table 1.4.

26

 

 

 

 

 

 

 

 

 

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CONVENTIONAL ANALYTICAL METHODS

The presence of trichothecenes as well as fumonisins in foods and
feedstuffs is potentially hazardous to human and animal health (Ueno, 1983;
CAST, 1989; Norred, 1993; Pohland,1993) and cause economic loss to farmers
and livestock producers (CAST, 1989). Therefore, elimination of the mycotoxins
from foods and feeds is very crucial. Although, prevention and elimination of
mycotoxins from human and animal diets is very difficult, detection and diversion
of contaminated raw materials from feed and food use can reduce the toxin in the
diets (CAST, 1989; Pestka et al, 1995). Conventional methods for mycotoxin
detection include thin layer chromatography (TLC), high performance liquid
chromatography (HPLC), gas chromatography (GC) and mass spectroscopy (MS)
(Pestka, 1994). Purification and detection of fumonisins will be reviewed in Part
II. In the following sections purification and detection of trichothecenes are

described.

Cleanup of trichothecenes prior to analytical detection.

Analyses of trichothecene mycotoxins in foods and feeds involve sample
extraction and a very extensive cleanup to purify the mycotoxins before detection
is made (Snyder, 1986; Chu, 1991; Gilbert, 1993). Based on trichothecene

solubility in various solvents, Snyder (1986) classified trichothecene mycotoxins

28

 

go:
go:
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29

into two groups. Group I toxins which have one or more acetyl groups, such as T-
2 toxin, HT-toxin, diacetoxyscirpenol, are very soluble in nonpolar solvent, such
as chloroform, methylene chloride, ethylacetate, diethyl ether, and acetone.
Group II toxins, which have no acetyl groups (e.g. DON, NW, and T-2 tetraol) are
highly soluble in polar solvents, such as ethanol, methanol, water, aqueous
methanol and aqueous acetonitrile because of stronger polar side groups in the
group II molecules. Therefore, nonpolar solvents are suitable for extraction of
group I trichothecenes, and polar ones for group II toxins (Snyder, 1986). When
a mixture of group I and II compounds is extracted, this author suggested using a
mixture of acetylacetate and acetonitrile as a solvent system. However, this
procedure requires an extensive purification to remove the acetylacetate from the
group I toxins. (Snyder, 1986).

Polar solvents such as methanol or acetonitrile in combination with water
are now commonly used for extraction of mycotoxins from sample matrices
because of increasing use of reverse-phase liquid chromatography column and
immunoassay (Whitaker et al., 1986). In addition, those solvent systems are less
toxic and less expensive than those used early. Furlong et al. (1995), for
example, used a mixture of methanol and aqueous 4% KCI solution at ratio of
9:1 (vol.lvol.) for extraction of trichothecenes from wheat. Meanwhile, a mixture of
acetonitrile and water at a ratio of 3:1 has also been used to extract DON, NIV
and zearalenone from cereal grains (Tanaka et al., 1985; 1986; 1988a,b; Park et

al., 1991; 1992; Muller and Schwadorf, 1993).

30

After extraction, trichothecenes can be separated from their crude extracts
via liquid/liquid or solid/liquid partitioning techniques (Snyder, 1986; Chu, 1991).
An example of a liquid/liquid partition solvent system used for T-2 toxin
separation is a mixture of methanol/ethylacetate/chIoroform at a ratio of 1:122
(vol.lvol.lvol.) (Hsu et al., 1972). A mixture of acetonitrile and petroleum ether at
a ratio of 1:1 (vol.lvol.) is also used in the separation of T-2 toxin or DAS
(Mirocha et al., 1976). In solid/liquid partition systems, columns which are
used for trichothecene cleanup include ferric gel (Mirocha et al., 1976), activated
charcoal (Morooka et al., 1972), activated charcoal-alumina (Romer, 1984), florisil
and silica gel (Tanaka et al., 1985), and charcoalzaluminazcelite at a ratio of 7:5:3
(wIw/w) (Eppley et al., 1986; Trucksess et al., 1986; 1987; Fernandez et al.,
1994). The availability of higher capacity and more effectively controlled size
absorption packing materials led to the development of a smaller but more
efficient column chromatography (Chu, 1991). Such smaller columns including
SAX (Thiel et al., 1991), Amberlite IRC 50 (Shepherd and Gilbert, 1986), Sep-Pak
(Mirocha et al., 1989) are now commonly used for mycotoxin purification, instead
of using large columns such as silica gel or using solvent partition methods (Chu,
1991). The following step after purification is mycotoxin detection which can be

performed with TLC, GC, HPLC, or MS methods (Pestka et al., 1995).

Detection of trichothecenes

TLC has always been a favorite method for analyses of mycotoxins

because of its simplicity and low cost (CAST, 1989). Most of the TLC studies

31
usually employ silica gel as an absorbent. The thickness of the thin layer is
generally 0.25 mm and various solvent systems are used as developing agents
(Ueno, 1983). Retention factor (Rf) values of some trichothecene mycotoxins are
summarized in Table 1.4.

Derivatization reactions are required when TLC is applied to
trichothecenes because most trichothecene toxins do not have useful absorption
or potential for fluorescence under ultraviolet or visible light (Ueno, 1983).
Compounds used as derivatization reagents include sulfuric acid (Ueno et al.,
1973; Gimeno, 1979), p-anisaldehyde (Scott et al., 1970), 4-(p-
nitrobenzyl)piridine (NBP) (Takitani et al., 1979), nicotinamide-Z-acetalpyridine
(Sano et al., 1982), and aluminum chloride (Romer, 1986; Trucksess et al., 1987;
Fernandez et al., 1994). Use of a certain derivatization reagent depends on the
objectives of trichothecene analysis, the kinds of trichothecene mycotoxins to be
analyzed, and the methods of toxin purification (Ueno, 1983). This author
suggested to use sulfuric acid, aluminium chloride, or nicotinamide-Z-
acetylpiridine for analysis sensitivity; aluminium chlon'de, NBP, or nicotinamide—Z-
acetylpin‘dine for toxin selectivity; sulfuric acid, aluminium chloride, or NBP for
procedure simplicity; aluminium chloride, or nicotinamide—2-acetylpiridine for
derivative stability. For general purposes, aluminium chloride or NBP is most
frequently used as a derivatization reagent (Ueno, 1983).

Color development using sulfuric acid, or p-anisaldehyde reagents is
performed by spraying the reagents on a TLC plate and then heating at 100-

130°C for about 20 minutes (Scott et al., 1970; Ueno et al., 1973). When sulfuric

32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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34

acid is used, group A and B trichothecenes generate a grayish black color, and a
brown color, respectively. The detection limit of this system is approximately 0.25
pg per spot (Ueno et al., 1973). In addition, under 360 nm UV light the group A
give a blue fluorescent color with a detection limit of 0.05 pg per spot (Ueno et al.,
1973). The same detection limits is also obtained when p-anisaldehyde is used as
a derivatization reagent, but group A trichothecenes give a pinkish violet color
and the group B trichothecenes yield a yellowish brown color(Scott et al., 1970).

Takitani et al. (1979) reported that a detection limit of 0.02-0.2 pg per spot
was possible by using NBP as a derivatization reagent. A blue-violet color for
trichothecenes was observed under long wave UV light after a TLC plate was
sprayed with NBP solution, heated at 150°C for about 30 minutes, cooled at room
temperature, and then dipped in tetraethylenepentamine solution. The blue-violet
color was a compound which was formed through N-alkylation of N-atom on the
piridine ring of the NBP reagent with the epoxy group of trichothecenes. These
investigators suggested that this method was applicable to all trichothecene
mycotoxins having a characteristic of 12, 13-epoxy group.

More sensitive and specific TLC methods were developed by Nelis and
Sensheimer (1981). They used nicotinamide as an alkylating reagent to produce
fluorescent derivatives. An epoxide-containing compound such as trichothecenes
is added to the solution of nicotinamide, a ketone (e.g. acetophenone), and
alcoholic KOH. A blue fluorescent color is developed by the addition of formic

acid at ambient conditions. These researchers reported that this system had a

35
detection limit of 0.1-2.0 ng per spot. If compared to NBP method, this technique
is better because it is approximately 100 folds more sensitive.

High-performance thin layer chromatography (HPTLC) has also been
applied to detection of DON, fusarenon-X, and NIV in cereal grains (Trucksess et
al. 1987). After purification, the toxins are spotted on a TLC plate which has
been previously impregnated with aluminum chloride. The plate is then
developed in two sequential solvent systems, firstly in chloroform:acetone:2-
propanol at ratio of 8:1:1(vol.lvol./vol.), and secondly in the same solvent mixture
but at a 14:3:3 ratio. After air drying, heating at 120°C for 8 minutes, and cooling
at room temperature, the plate is observed under long-wave UV light. The toxins
appear as blue fluorescent spots. Rf values of DON, fusarenon-X, and NIV are
0.5, 0.4, and 0.1 respectively. Average recovery of these three toxins added to
cereal grains at levels of 100 and 200 ppb was 83%, and the detection limit of this
system was 50 pbb (Trucksess et al., 1987). Fernandez et al. (1994) also used
this technique for detection of DON in 1991 US. winter and spring wheat and
they found that the limit of determination was 40 ppb, and average recoveries of
DON-spiked wheat samples at levels of 200, 400, and 800 ppb were 83, 82 and
72%, respectively.

Trichothecenes can be analyzed either individually (Lauren and
Greenhalg, 1987) or collectively (Lauren and Agnew, 1991) with HPLC methods.
Lauren and Agnew (1991) developed a multitoxin screening method for Fusarium
mycotoxins using HPLC techniques. They hydrolyzed trichothecenes (DON,

NIV, scirpentriol, and T-2 tetraol) to form the toxin parent alcohol. This parent

36

alcohol is then detected with HPLC using a longwave UV detector. Recoveries of
parent alcohol spiked into wheat and maize extracts at a level of 0.5 ppm ranged
from 55 to 103%. Levels of 50 ppb or less were detectable by this method.
Meanwhile, Lauren and Greenhalg (1987) observed a detection limit of 50-100
ppb for DON and NIV without hydrolysis. A higher detection limit (up to 0.02 ppb
for DON and NIV) was achieved when HPLC was equipped with a fluorescent
detector (Gilbert, 1991). This system involves post column heating with alkali to
generate formaldehyde which is then derivatized with methyl acetoacetate and
ammonium acetate in a second reaction coil (Gilbert, 1991).

Trichothecene have frequently been analyzed by GC methods (Ueno,
1983; Snyder, 1986). GC analysis require derivatization of trichothecenes to form
either trimethylsilyl (TMS) or heptafluorobutyril (HFB) derivatives, prior to
detection with a capillary column which is equipped with a flame ionization
detector (FlD) (Kamimura et al., 1981; Trucksess et al., 1987) or an electron
capture detector (ECD) (Ware et al., 1984). Detection limits of GC—FID are 200
ppb and 100 ppb for group A and B trichothecenes, respectively (Kamimura et al.,
1981). Detection limits of approximately 80 ppb for group A trichothecenes and 2
ppb for group B trichothecenes are achieved when ECD-GC is used (Ware et al.,
1984). GC analyses are generally preferred to HPLC methods because GC has
a greater sensitivity and specificity. The greater separating capacity of GC
compared with that of HPLC also enables researchers to monitor a greater

number of trichothecenes in the same extracts. In addition, 60 when coupled

 

a q:
sou;
met
M8
the
Clea

GC

37
with MS analysis can simultaneously detect and confirm trichothecene
mycotoxins in a food or feed sample (Gilbert, 1993).

In earlier investigations, MS detection methods were not considered to be
a quantitative analytical tool for mycotoxins. However, a high resolution MS
coupled with computer search and integration capabilities has led to new MS
methods for both identification and quantification of mycotoxins (Plattner, 1986).
MS methods are preferable for trichothecene analyses since they do not require
the compounds to have a chromophore (Chu, 1991). In some cases, extensive
clean-up steps are not necessary for MS and a detection limit of tandem
GCIMSIMS systems is 1 ppb for T—2 toxin in urine and blood (Mirocha, et al.,
1989)

In summary, analysis of trichothecene mycotoxins using a TLC method are
rapid, simple and low cost. This method, however, is generally low in sensitivity
and selectivity (Bamburg, 1983; Ueno, 1983). Higher sensitivity and selectivity of
trichothecenes analysis can be achieved by using GC, HPLC, and MS techniques
although they require laborious sample cleanups, and expensive instruments

(Pestka, 1988; 1994; Chu, 1991).

IMMUNOASSAYS FOR FUSARIUM MYCOTOXINS

Fusarium mycotoxins are a group of secondary metabolites produced by
toxigenic strains of Fusarium including Fusan‘um monilifomie that produces
fumonisins and Fusarium gramineamm that produces DON (Betina, 1989; Bacon
and Nelson, 1994). Fusan'um mycotoxins especially DON and fumonisins are
toxic to both human and animals and commonly contaminate cereal and cereal
products (Bamburg, 1983; Tanaka et al., 1988b; Marasas et al., 1988a;
Sydenham et al., 1991). Elimination of mycotoxins from human and animal foods
is commonly accomplished via detection and diversion mycotoxin-contaminated
raw materials from feed and finished food use (Pestka, 1994). Mycotoxin
detection can be carried out using either conventional methods such as TLC, GC,
HPLC and MS methods or immunoassay techniques such as ELISA. ELlSAs are
commonly preferred to conventional methods because of simplicity, rapidity, and
applicability both in the field and laboratory (El-Nakib et al, 1981).

The basis of mycotoxin immunoassays involves competition between a
free mycotoxin and a labeled mycotoxin for an antibody binding site (Pestka,
1988). The purpose of the following review is to discuss Fusarium-mycotoxin
immunoassays with specific emphasis on antibody production, mycotoxin
immunoassay format, application of mycotoxin immunoassays in foods, and

commercial mycotoxin immunoassay kits for foods.

38

 

 

rx

39

Antibody production

Antibodies or immunoglobulins are glycoproteins that are produced and
secreted into the body fluids by B lymphocytes of animals in response to a foreign
chemical (antigen) significantly different from the animal’s own chemical
(Candlish, 1991). lmmunogenicity refers to the capacity of an antigen to induce
an animal’s immune response and usually dependent on its chemical structure
and its size (Pestka et al., 1995). Production of a highly specific antibody in an
animal is mainly influenced by the type of antigens, dose and route of
immunizations, the number and types of accessory cells such as macrophages
that initially interact with antigen and induce lymphocyte activation, and the nature
of responding lymphocytes (Abbas et al., 1994). Generally, immunogen must be
degradable and must have an epitope that can bind to the cell-surface antibody of
a B virgin cell. It must also have at least one site that can be recognized
simultaneously by a class II protein and by a T-cell receptor, thus facilitating cell-
to-cell communication between helper T cells and B cells can occur (Harlow and
Lane, 1988). Compounds smaller than 3000 daltons (hapten) may be able to
bind to the surface antibody of B cells, but may not have suitable site for the 3
simultaneous binding of a class II protein and a T-cell receptor.

Most Fusarium mycotoxins are of low molecular weight (250-500 dalton)
(Betina, 1989), and thus are not immunogenic by themselves (Chu,'1986). This
problem can be overcome through conjugation of the toxins to larger

immunogenic molecules (carriers).

4O

Mycotoxin conjugation. Mycotoxin conjugation is a reaction of a
mycotoxin and a carrier protein to form a mycotoxin-protein carrier conjugate so
that the mycotoxin becomes immunogen. Carrier molecules that are commonly
used for mycotoxin conjugation are capable of imparting immunogenicity to
covalently coupled mycotoxins or haptens. These include bovine serum albumin
(BSA), chicken ovalbumine (OA), and keyhole limpet hemacyanin (KLH), and
cholera toxin (CT) (Chu, 1986; Harlow and Lane, 1988).

BSA is very soluble and a good protein carrier (Harlow and Lane, 1998).
These authors reported that BSA has 30-35 lysines, 19 tyrosines, 35 cysteines,
39 aspartic acids, and 59 glutamic acids that are available for coupling.
Meanwhile, CA has 20 lysines, 10 tyrosines, 6 cysteines, 14 aspartic acids, and
33 glutamic acid residues.

KLH (MW 4.5x 105 to 1.3x107), a copper containing protein, belongs to a
group of non—heme proteins called hemacyanins which are found in arthropods
and molluscs (Senozan et al., 1981). KLH is isolated from the mollusc Megathura
creulata. ln Tris buffer, pH 7.4, this protein exists in 5 five states of aggregation
which have sediment coefficients of 102$, 130$, 150$, 1708, and 1868
(Senozan et al., 1981; Herckovits, 1988). It will reversibly dissociate to subunits
when pH changes moderately. Both lowering and raising of pH will cause
dissociation (Herckovits, 1988) and at pH 8.9 it will completely dissociate to
subunits. Each subunit contains oxygen binding sites which can bind to two

copper atoms per molecule oxygen in KLH (Senozan et al., 1981; Herckovits,

41
1988). Unlike BSA, KLH is sometimes likely precipitated during coupling due to its
large size, and this can make difficult in some cases (Harlow Lane, 1988).

Cholera toxin (CT) (MW 86,000) is a protein exotoxin produced by Vibrio
cholerae and has been shown to have strong and oral systemic adjuvant
properties when co-administered with unrelated antigen (Liang et al, 1978; Lycke
and Holmgren, 1986). CT consists of two types of subunits, a single ’heavy’
subunit of molecular weight 28,000 which noncovalently attached to a 58,000-
MW aggregate of ’Iight’ subunits (Holmgren, 1981). CT has several
advantages when used for generating antibodies (Azcona-Olivera et al., 1992a;
Abouzied et al., 1993). Firstly, the procedure was rapid and yield a good quality
of antibodies than standard protocols when the toxin was applied to generate F B1
antibody. Secondly, CT might be an humane alternative to Freund’s adjuvant
since abscesses, ulcers, or granulomas at injection sites which usually appeared
after F reund’s adjuvant immunization, are not observed after CT injection. Thirdly,
because low doses of CT-immunogen yields a rapid and strong antibody
response, CT would be valuable when mycotoxin availability is limited.

Conjugation of a mycotoxin to a protein carrier can be achieved following
chemical reaction of functional groups present on the mycotoxin with functional
groups present on the carrier (Chu, 1986). Mycotoxins which already contain
reactive groups such as fumonisin 81 , 32, and 83 can be coupled directly to
protein molecules by using glutaraldehyde as a protein linker (Figure 1.5)
(Azcona-Olivera, 1992a,b; Fukuda et al., 1994; Usleber et al., 1994). However,

generation of protein conjugates for mycotoxins which do not contain functional

42
groups such as DON (Casale et al., 1988) is much more complex because a
reactive group has to be introduced by chemical synthesis to the toxins prior to
the conjugation reaction (Figure 1.5).

A reactive group commonly introduced to trichothecenes is carboxylic acid
which is performed by reacting the toxins with bifunctional acid anhydrides, such as
succinic or glutaric anhydrides at the presence of a catalyst such as 4,N,N-
dimethylaminopyn'dine or pyridine. Hemisuccinate and hemiglutarate of T2 toxin
(Chu et al., 1979), diacetylscirpenol (Chu et al., 1984a), 3-acetyl DON (Kemp et
al., 1986; Usleber et al., 1991), and DON (Usleber et al., 1993), as well as
hemisuccinate of DON triacetate (Zhang et al., 1986), DON (Casale et al., 1988;
Usleber et al., 1991), and 4, 15-diacetylnivalenol (Abouzied et al., 1993) have
been prepared prior to the conjugation of the toxins to protein carriers.

Conjugation of hemisuccinic or hemiglutan'c trichothecenes to protein carriers
is usually accomplished using a mixed-anhydride (MA) or an activated ester (AE)
method. In MA method the trichothecene derivatives are generally activated to their
corresponding anhydride by isobutyl chloroforrnate in dry tetrahydrofuran and
triethylamine and simultaneously coupled with protein (Gendloff et al., 1986); where
as, in the AE method, N-hydroxysuccinimide and dicyclohexylcarbodiimide in
dimethylforrnamide solution are used to activate the derivatives prior to conjugation

process (Kitagawa et al., 1981).

43

 

 

 

 

I
3‘“; H non-om

:10
m1. (‘ q 1”

 

 

  

. ' g
" our ir-cl,-(cu,),~¢'|;“l""“" {.

O—BSA

 

 

DON-E3834

Figure 1.5. Preparation of immunogen for (left) fumonisins and (right)
deoxynivalenol (DON) (Pestka et al., 1995)

44

Polyclonal antibodies. After successful production, characterization, and
purification of a mycotoxin-protein conjugate, suitable animal species are then
immunized with the purified conjugate (Chu, 1986; Harlow and Lane, 1988). These
latter authors suggest the use of rabbits, goats, or sheep for polyclonal antibodies
and mice for monoclonal antibodies because only mice which have tumor cell lines
for the efficient fusion of plasma cells.

Multiple-site intracutaneous immunizations of rabbits with 100—1000 pg of
mycotoxin-protein conjugate which has been mixed with an adjuvant are commonly
performed to generate antibodies (Hariow and Lane, 1988). The conjugate is usually
emulsified with an oil-base adjuvant containing killed Mycobacten’um such as
“complete” Freund’s adjuvant at a ratio of 1:1 (vol/vol.) to allow slow release of the
immunogen and nonspecifically induce immune response (Usleber, 1994).
“Complete” Freund’s adjuvant is used for primary immunization and “incomplete” (not
containing killed Mycobacten’um) one for subsequent injections which are usually
given in 2-4 week intervals. High titer rabbit antisera commonly can be generated as
early as 4 weeks after the initial injection (Zang et al., 1986; Wang and Chu, 1991;
Abouzied et al., 1993; Usleber et al., 1993; 1994).

Rabbit antisera contain antibodies which are generated by multiple B-cell
clones and vary in specificity; therefore, they are considered polyclonal (Pestka et
al., 1995). A major advantage of polyclonal antibodies is that they are easy and
inexpensive to produce, and often contain a subclone of high affinity antibody

(Usleber et al., 1994). However, the quality of polyclonal antisera in terms of affinity,

45
specificity as well as physical and chemical stability varies from bleeding to bleeding
and from rabbit to rabbit (Pestka et al., 1995). As a consequence, use of these
antibodies for constniction of commercial kits with defined performance
characteristics is difficult. This difficulty can be eliminated when monoclonal

antibodies are used.

Monoclonal antibodies. Production of monoclonal antibodies involves
animal immunization and fusion of splenocytes with a tumor cell line (Kohler and
Milstein, 1975). lntraperitoneal or subcutaneous immunizations of female BALBIc
mice with 5-50 pg mycotoxin-protein conjugate per injection are generally used to
induce B cells which an synthesize the mycotoxin-specific antibodies (Azcona-
Olivera, 1992b; Fukuda et al., 1994). The immunizations are usually given three
times at two weeks intervals. The first and second injections were with an emulsion
of adjuvant and the conjugate, and the final one was with the conjugation without
adjuvant. Four days later, the mice producing mycotoxin-specific antibodies are
killed and their spleen cells which contain high numbers of antibody-secreting B
A cells are fused with myeloma cells (mouse cells which have all cellular processes
necessary for the secretion of antibody but have no ability to produce antibody)
using polyethylene glycol (Galfre and Milstein, 1981).

During fusion, only about 1% of the starting cells are fused and only 1 in 105
form viable hybrids (Harlow and Lane, 1988). In tissue cultures the cells from
immunized mouse spleens can not grow but the unfused myeloma cells can adapt

and grow well (Harlow and Lane, 1988). These unfused cells have to be eliminated,

46

so they do not block the growth of the hybrid cells. Unfused myeloma cells, in which
the hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) in the salvage
nucleotide synthesis pathway has been mutated, are killed by the addition of
hypoxanthine, aminopterin and thymidine to the culture media (Azcona-Olivera,
1992b). Aminopterin will block de novo nucleotide synthesis pathway and force
every cell in the culture to synthesize its purine nucleotides via salvage pathway.
Under these conditions, cells containing a non functional HPRT protein such as
myeloma cells will die and hybrids between spleen B cells with a functional HPRT
and myeloma cells with a non-functional HPRT will continue to grow by using
hypoxanthine and thymidine for producing their nucleotides (Hariow and Lane,
1988)

Supematants of the hybridoma cultures can be screened for the presence of
the mycotoxin-specific antibodies by competitive indirect enzyme-linked
immunosorbent assay (Cl-ELISA) (Azcona-Olivera et al., 1992b). The selected
cultures are then successively scaled up and cloned by limiting dilution at 0.5-1
cellMell (Goding, 1980). An immortalized subclone that consistently secretes
antibody of desired affinity, specificity, and performance characteristics is then '
expanded to produce a large amount of monoclonal antibodies. Although
monoclonal antibody generation is expensive, time consuming and requires for
tissue culture facilities, these antibodies tend to exhibit a low degree of interassay
Variation and give highly reproducible results (Pestka et al., 1995). Therefore, they

are suitable for use in the development of commercial kits.

47
Recombinant antibodies. Beside using hybridoma technology, antibodies
can be produced through gene recombinant technology which was developed
recently. The gene technology involves reanangement or assembly of germ line V—
genes, surface display of antibody, affinity selection, affinity maturation and soluble
antibody production (Figure 1.6, Winter et al., 1994).

- Initially antibody genes were taken from hybridomas, cloned into plasmid
vectors and expressed as fragments in bacteria (Better et al., 1988) or as complete
antibodies in mammalian cells (Oi et al., 1983). Later, Orlandi et al. (1989) isolated
antibody genes directly form lymphocytes of immunized animals, and then
propagated the genes by using universal primers and polymerase chain reaction
(PCR) prior to expression the genes in bacteria. In addition, by building restriction
sites into these primers, the amplified DNA can be cloned directly for expression in
bacteria (Milstein, 1990). Like hybridoma technology, the later approach still relies
on animal immunization to generate antigen specific lymphocyte cells. The
hybridoma technology an immortalize these cells (Kohler and Milstein, 1975), while
the gene technology can immortalize their genes (Milstein,1990). In future, it may
become possible to generate antibody without animal immunization. Antibody genes
are taken from unimmunized animal, and then amplify using universal primer and
PCR (Orlandi et al., 1989). Prior to expression in bacteria, the genes can be readily
manipulated by cutting and pasting of restriction fragments (Milstein, 1990) or by site

directed mutagenesis (Better et al., 1983) to construct a new and desired antibodies.

48

Unrearranged V-genes
‘ Steps 1.2 Steps 1,2 ‘

   

Rearranged V-genes Assembled V-genes in phage(mid)
‘ Step 3 5‘99 3 *

 

 

Figure 1.6. Process of antibody production in vivo (left) and using gene technology
(right). Step 1: rearrangement or assembly of germ line V-genes; step
2: surface displaying of antibody; step 3: antigen-driven or affinity
selection; step 4: affinity maturation; step 5: production of soluble
antibody or antibody fragment (Adapted from VWnter et al., 1994).

49

Mycotoxin immunoassay format

A number of immunoassay formats have been developed for mycotoxin
analysis. Initially, competitive radioactive immunoassays have been developed
for mycotoxins, at which a mixture of a constant amount of a radiolabeled
mycotoxin and mycotoxin standards or unknown samples is incubated with
specific antibodies (Pestka et al., 1995). Radioactivity of unbound radiolabeled
mycotoxins in the solution is then measured after a complex of antibodies and
radiolabeled toxin is removed from solution. Amount of the toxin in a sample is
reversely related with unbound radiolabeled in the solution. Because of inherent
problems with radioactive reagents, enzyme-linked immunosorbent assays
(ELISAS) were subsequently developed on the procedures of Engvall and
Perlman (1971).

Solid phase supports used for mycotoxin ELISA include microtiter plates,
beads, tubes, and dipsticks (T archa, 1991). These solid supports are most
commonly made from polystyrene because of its low cost, easy process, optical
clarity and high-protein binding (Tarcha, 1991). Among solid phase supports,
polystyrene microtiter plates have been most commonly used because of
supporting technology, such as removable stn'ps, multiwell pipettes, automated
washers, and spectrophotometers (Pestka, 1994).

ELISA procedures commonly applied for mycotoxin detection are
competitive direct ELISA (CD-ELISA) and the Cl-ELISA (Figure 1.7). In the CD-

ELISA, an enzyme-labeled mycotoxin and a free mycotoxin from samples are

5O

incubated together over a solid phase-bound mycotoxin antibody (Pestka, 1988).
This assay is based on the competition between free and the labeled mycotoxins.
A concentration of mycotoxin in sample is inversely related to a complex of
enzyme-labeled mycotoxin and the bound antibody. After the addition of an
appropriate enzyme substrate, the bound antibody as well as sample mycotoxin
concentrations can be calculated based on the intensity of color development
which is measured spectrophotometrically. In the Cl—ELISA, a solid-phase
mycotoxin-protein conjugate competes with a free mycotoxin for binding to a
soluble-mycotoxin specific antibody. A second antibody which has been labeled
with an enzyme is then used to determine total bound antibodies and free
mycotoxin concentration.

Recently, Pestka (1991) developed immunoassay format, called
ELISAGRAM, by combining the sensitivity and selectivity of competitive ELISA
with the capability of high-performance thin-layer chromatography (HPTLC) to
separate structurally related mycotoxins (Figure 1.8). ELISAGRAM involves
coating nitrocellulose (NC) membrane with mycotoxin-specific monoclonal
antibodies, separation of mycotoxins by HPTLC, blotted the HPTLC with the NC,
incubation of NC with mycotoxin-enzyme conjugate to identify mycotoxins bound
to the monoclonal antibodies, detection of bound enzyme conjugate with a
precipitating substrate, and visual or densitometric assessment of inhibition bands
indicative of cross-reacting mycotoxins. This format has successfully been used
to detect zearalenones and aflatoxins, and the result is similar to the competitive

ELISA (Pestka, 1991). A major advantage of this procedure is that detection and

 

 

 

 

Direct “ , {0%
.-‘ $0,
0

 

 

 

 

 

Figure 1.7. Competitive ELISA for mycotoxins (adapted from Pestka, 1988). In
direct ELISA, enzyme-labeled mycotoxins (ENZ) are simultaneously
incubated with free mycotoxins (U)over solid-phase bound antibodies
(AB). Concentration of free mycotoxins is inversely related to bound
enzyme-labeled mycotoxins and can be measured quantitatively
using spectrophotometer after color development by the addition of
enzyme substrate. In indirect competitive ELISA, mycotoxin specific
antibodies (AB) compete with free mycotoxins (D) to solid-phase
mycotoxin-carrier protein (CP) conjugate. Second antibodies (ab-
ENZ) which have been labeled with enzyme are then added to
determined total antibody bound. Concentration of free toxin is
inversely related to bound enzyme-labeled antibodies.

52

 

 

1. SEPARATE HAPTENS ON
CHANNELED
HPTLC SILICA GEL PLATE (I)

 

 

 

 

 

a IMMUNOSPECIFIC TRANSFER:
FILTER PAPER (field
ANTIBODY-GOA NC (-)

 

 

 

HPTLC (-)

' 3' $503330». HRP
m ’
OONJUGATE (8)

4. INCUBATE NC WITH
PRECIPITATING HRP
SUBSTRATE. IDENTIFY
HAPTENS AS
INHIBIT ORY BANDS

 

5. PERFORM SCANNING
DENSITOMETRY

:1' r5: ‘0." " -‘ - 1,-2.
. I," D A . .3
. ~ - ~ , c
.‘..L " ,' -, a
.-]a' c.’ . 2 ' ‘ .0
. .‘ . "\r' 9_ .193. .
': 0,“. ‘x i. -‘.- vr ..
A' ' ’2‘. ."f'; .~ "' .-
. ' " . t . .0 .' . D
.:‘ ‘.? ‘.‘ :3. t : ’3‘ ‘0
,' '. ‘0. a." 0. I ‘ z" ‘. ‘3‘. |
9. ..~‘._.. “a" Q .'.
. ' ‘5. 0", . ‘. .0. “
. . u .
. u- '0. . ¢ . .
'0; O a. ' .0 a _'
I ' I f f
A. .
K ‘ .

 

 

 

Figure 1.8. ELISAGRAM procedure for mycotoxins (Pestka, 1991).

53
confirmation of multiple haptens can be performed simultaneously with a single
cross-reactive antibody.

More recently, a Computer—Assisted-MuItianalyte Assay System (CAMAS)
has been devised by Abouzied et al. (1993) for simultaneously detection of
fumonisins, aflatoxins and zearalenones. In this format, monoclonal antibodies
for each of the toxins are immobilized as multiple lines on nitro cellulose
membrane strips and sectored into hydrophobic compartments to minimize
reagent use. A modified ELISA is performed at which free mycotoxins and
enzyme-labeled mycotoxins compete for binding to the nitrocellulose-bound
antibodies. By addition of a precipiting substrate, line-color intensity which is
inversely related to mycotoxin concentration is developed. The color intensity
can be measured quantitatively using a camera, video monitor, and
microcomputer equipped with a video digitizing board. An advantage of this
approach is that detection of several mycotoxins man be performed
simultaneously in less than 30 minutes (Abouzied et al., 1993). In addition, assay
data can be recorded in the minicomputer hard disk.

Other immunochemical formats called “hit and run” assay for T-2 toxin
(Warden et al., 1987) and immunochromatography for group A trichothecenes
(Chu and Lee, 1989) have been also developed. In the “hit and run” assay, a T2
toxin column is equilibrated with fluoresce isothiocyanate (FITC)-Iabeled Fab
fragments of lgG T-2 toxin antibodies. Samples containing T-2 toxin are injected
to the column so the toxin binds to FlTC-Fab fragments. After washing, FITC-

Fab—T-2 toxin complexes are eluted and then the toxin is determined in a

54
standard flow through fluorometer (Warden, et al., 1987). In the
immunochromatography approach, which is a combination of HPLC and ELISA
methods, group A trichothecenes are separated on C"; reversed-phase column.
Individual fractions eluted from the column are assessed by ELISA using
mycotoxin-specific antibodies. This approach can both identify and determine the
concentration of individual group A trichothecene and its detection limit is 2 ng

(Chu and Lee, 1989).

Application of mycotoxin immunoassays in foods

Use of immunoassay techniques for mycotoxin detection in foods and
feeds is generally preferred to conventional methods, such as TLC, HPLC, GC,
or MS because of lower costs, easier execution and quicker result (Pestka,
1988). Antibodies against important Fusarium mycotoxins such as fumonisins,
trichothecenes, zearalenones have been produced and used for development of
radioactive immunoassay (RIA) and ELISA methods. Both methods have been
successfully used for mycotoxin screenings in a diverse array of foods and feeds.
Selected examples of reported immunoassay for Fusarium mycotoxins are
summarized in Table 1.6.

An aqueous system is required for immunoassays because antibody and
enzyme conjugate activities. Originally, extraction by standard procedures,
evaporation, and reconstitution in aqueous buffer have to be done prior to
mycotoxin analysis in solid foods such as grain and grain products (Pestka,

1988). In some cases, a column clean up is also performed to obtain higher

55

Table 1.6. Selected Fusarium mycotoxin immunoassays in foods

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Toxin Format Food analyzed Detection Reference
limit (ppb)
Fumonisin B, ELISA Buffer solution 50.0 Azcona-Olivera et al.,
1992a,b
ELISA Feed 250.0 -”-
ELISA Buffer solution 0.6 Usleber et al., 1994
ELISA Corn 10.0 -”-
ELISA Buffer solution 100 Fukuda et al., 1994
ELISA Foods 200 Pestka et al., 1994
Trichothecenes:
Acetyldeoxynivalencl ELISA Rice 1.0 Kemp et al., 1986
15~Acetyldeoxy- ELISA Wheat 50.0-100.0 Usleber et al., 1993
nivalenol
4,15-Diacetyl- ELISA Buffer solution 5.0 Abouzied et al., 1993
deoxynivalenol
Diacetoxyscirpenol ELISA Culture 16.0 Hack et al., 1989
DON RIA Corn, wheat 20.0 Xu et al., 1986
ELISA Corn 200.0 Casale et al., 1988
ELISA Grain-based 1000.0 Abouzied et al., 1991
food
ELISA Buffer solution 1.0 Usleber et al., 1991
Nivalenol tetraacetate RIA Buffer solution 5.0 Wang and Chu, 1991
Roridin A ELISA Feed 5.0 Martbauer et al., 1988
T-2 toxin RIA Corn, wheat 0.1 Lee and Chu, 1981 a
RIA Milk 2.5 Lee and Chu, 1981b
ELISA Corn 50.0 Gendloff et al., 1984
ELISA Corn, wheat 2.5 Pestka et al., 1981a
ELISA Milk 0.2 Fan et al., 1984
ELISA Wheat 0.5 Chiba et al., 1988
ELISA Cereal grains 100 Vetro et al., 1994
Zearalenone ELISA Corn 1.0 Warner and Pestka,
1986
ELISA Grain-based 2.5 Warner and Pestka,
food 1987

 

ELISA and RIA indicate enzyme-linked immunoassay and radioimmunoassay, respectively.

 

56

sensitivity (Pestka et al., 1981a). However, since mycotoxin-horseradish
peroxidase conjugate as well as solid-phase bound antibodies remain stable in
35% (vol/vol.) methanol in water (Ram et al., 1986), mycotoxin immunoassays
can be directly applied to crude extracts of food samples (Usleber et al., 1994).
In a liquid system, such as milk, mycotoxins can be detect directly with
immunoassay approach although cleanup with a Sep—Pak or affinity column can
increase the detection limit of immunoassays (Pestka et al., 1981 b).

Sample matrix interference should be considered carefully when detecting
mycotoxins in foods or feeds using immunoassay techniques (Laamanen and
Veijalainen, 1992). These authors reported that certain substances in food
sample might affect color development. When they detected T-2 toxin in millet
grains with competitive ELISA assays, 75% of the grain gave higher optical
density (OD) values than the negative control. However, a lower OD value than
the negative control was obtained when the system applied to fermented foods,
processed foods or feed stuffs. This problem could be eliminated by retesting the
samples at higher dilution (Laamanen and Veijalainen, 1992). Incorporating
toxin-free sample extracts during standard curve preparations can also minimize
the false positive or negative reactions (Ram et al., 1986). Since reaction
between antibodies and antigens occurs optimally at neutral pH, sample pH must

also be considered prior to mycotoxin immunoassays (Pestka et al., 1995).

57

Commercial mycotoxin immunoassay kits for foods

Extensive research in ELISA has been performed since its discovery in
1971 (Engvall and Perrnann, 1971) and the results demonstrate that ELISA
methods are feasible and reliable for mycotoxin analysis in foods and feeds (Chu,
1986). As a consequence, a number of immunoassay kits have been developed
and marketed in the United States as a tool for monitoring mycotoxins in foods
and feeds (Table 1.7). Generally, commercial immunoassay kits have worked
well both in laboratories and in the field (Azer and Cooper, 1991; Domer et al.,
1993). Some have been evaluated and approved by several professional
organizations including Association of Official Analytical Chemists (AOAC)
International (Park et al., 1989 a,b). To facilitate evaluation and certification of
rapid test kits used for safety screening of foods, the AOAC Research Institute
has been set up recently (Pestka et al., 1995). In October, 1992, this
organization signed a Memorandum of Understanding with the US. Department
of Agriculture’s Federal Grain Inspection Service (FGIS) concerning the Test Kit
Performance Testing Program (Pestka, et al., 1995). Aflatoxin test kits have
been evaluated using FGIS protocols and certified to claim “Performance Tested
in Accordance with Standards Established by FGIS for Test Kits to Detect
Aflatoxin Residues in Grain and Grain Products”. More recently, two DON
ELISAs have been similarly certified by FGIS.

Although commercial immunoassay kits have been certified by FGIS, they

must be evaluated critically prior to adopting the systems. Criteria for adoption of

58

 

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60
a mycotoxin immunoassay include: detection limits and sensitivity ranges,
requirements for rapid screening and/or quantitation, mycotoxin specificity,
effectiveness of recommended extraction procedures, field stability, inter-and

intra- assay reproducibility (Pestka, 1988).

61

PART II

DEVELOPMENT AND APPLICATION OF AN ELISA FOR FUMONISIN 81
IN FUNGAL CULTURES, CORN AND CORN PRODUCTS

ABSTRACT

DEVELOPMENT AND APPLICATION OF AN ELISA FOR FUMONISIN 81 IN
FUNGAL CULTURES, CORN AND CORN PRODUCTS

By

Sutikno

Mouse monoclonal antibodies, rabbit and sheep polyclonal antibodies against
fumonisin B1 (FB1 ) were readily generated after immunization with keyhole limpet
hemacyanin (KLH) as a protein carrier. When these antibodies were used in
competitive inhibition enzyme-linked immunosorbent assay (ELISA), the sheep
antisera had the highest affinity and were the most specific to FB1 as compared to
the mouse monoclonal and rabbit polyclonal antibodies. Cross reactivities of these
sheep antisera toward fumonisin B1 , Bz , and 83 were 100, 24 and 30%,
respectively. When competitive direct ELISA (CD—ELISA) employing these FB1
sheep antisera was used to detect F31 in Fusarium corn culture, corn and com
products, it yielded approximately two fold higher FB1 levels than high performance
liquid chromatography (HPLC). Its perfomance was much better than a monoclonal
antibody based CD—ELISA that was previously developed in our laboratory. This
CD-ELISA had a strong positive relationship with HPLC, suggesting that it would be

useful for screening human and animal foods from fumonisins.

62

INTRODUCTION

Background.

Fumonisins are toxic secondary metabolites, found worldwide that are
produced by Fusarium monilifonne and F. pmliferatum. These toxins are
elaborated by the fungi in corn in the field and during storage (Bezuidenhout et al
1988; Gelderblom et al., 1988a). Six different fumonisins, fumonisin A1, A2, B1,
82, 33, and B4, have been chemically characterized(Figure 1.2) (Bezuidenhout et
al., 1988; Cawood et al., 1991; Plattner et al., 1992). Fumonisin 8, (F81) is the
most toxic and primary fumonisin produced by Fusarium moniIifonne (Gelderblom
et al., 1988b). F81 produces characteristic effects in different species, including
hepatic cancer in rats, brain lesions in horses (equine leukoencephalomalacia,
ELEM), and pulmonary lesions in pigs (porcine pulmonary edema, PPE).

Vifilson et al. (1985) observed hepatic cancer in rats that were fed with
corn naturally contaminated with the Fusarium moniliforme for extended periods
(up to 176 days). Marasas et al. (1984b) also found liver tumors in rats by
feeding Fusarium monilifonne culture materials. Gelderblom et al. (1988b)
performed experiments to investigate the toxic metabolite of Fusarium
moniliforme. Through chromatographic procedures, these investigators identified
the water-soluble fraction had potential tumor promoting capacity. Bezuidenhout

et al. (1988) and Gelderblom et al. (1988b) isolated fumonisin compounds from

63

64
the water-soluble material and determined their structure. Similarly, Voss et al.

(1989) developed a rat bioassay for hepatotoxic lesions that were produced by
feeding Fusan‘um monilifonne cultures or naturally contaminated corn. They
found that a water-extractable, rather than a chloroform-extractable one, was a
causal agent of both hepatocellular carcinoma in rats and brain lesions in horses.

ELEM is a neurotoxic disease that occurs only in Equidae and is
characterized by multifocal liquefactive necrosis of cereme white matter (Marasas et
al., 1988b). ELEM has also been demonstrated in separate experiments in which
horses were administered purified FB1 either orally (Kellerman et al., 1990) or
intravenously (Marasas et al., 1988b). Clinical signs begin with lethargy, head
pressing and no appetite, and over a period of days progresses to convulsions and
death (Ross et al., 1991). Other signs of intoxication are liver damage and altered
serum sphingosinezsphinganine ratios (Wang et al., 1991). Plattner et al. (1990)
identified F81 in extracts obtained from hepatotoxic and ELEM cases.

PPE is an unusual disease in pig, which is characterized by recumbency and
death (Norred and Voss, 1994). Outbreaks of PPE have occurred in Georgia
(Colvin and Harrison, 1992), and several Midwestern states (Osweiler et al., 1992;
Ross et al., 1992). These outbreaks were associated with the consumption of corn
screenings that were contaminated with F. monilifonne (Harrison et al., 1990). When
purified F81 is injected intravenously into pigs at a dose level of 0.40 mglkg body
weight for 4 days, pigs die by day 5 and have lung edema and hydrothorax similar to
pigs diagnosed with PPE. PPE does not occur when pigs are either injected with a

smaller dose of F31 (0.17 mglkg body weight) or with FBz at a dose level of 0.30

65
mglkg body weight (Harrison et al., 1990). These observations strongly suggest that

F81 is a causative agent of PPE. This finding has been confirmed by other
investigators by feeding pigs with com contaminated with FB1 (Osweiler et al., 1992;
Riley et al., 1993). Hascheck et al. (1992) reproduced PPE in pigs by injection of
purified FB1. These investigators suggested a possible mechanism for induction of
PPE where by F81 disrupts sphingolipid biosynthesis in the liver. This leads to
damaged hepatocyte membranes, which are released into the bloodstream. These
membrane fragments are phagocytized by pulmonary intravascular macrophages
which then release enzymes and other mediators wpable of increasing mpillary
permeability in the lung with subsequent edema. The fact that PPE has not been
found in other species, perhaps because of much lower levels of pulmonary
macrophages in other species (Winkler, 1988).

Correlations between Fusarium monilifonne food contamination and
human esophageal cancer have been reported in South Africa (Marasas et al.,
1988a) and in China (Cheng et al., 1985) suggesting that fumonisins are a
possible etiologic agent for this disease. Sydenham et al. (1990) surveyed corn
from areas with high and low rates of human esophageal cancer in Transkei,
South Africa. Samples taken from the high rate cancer area had higher levels of
Fusarium monilifonne and fumonisins. The authors suggested that consumption
of corn contaminated with Fusan‘um moniliforme may be a factor in the high rate

of human esophageal cancer.

66
Analytical methods

Commonly used methods for detecting and quantitating fumonisins are thin
layer chromatography (TLC), high performance liquid chromatography (HPLC),
and mass spectroscopy (MS). Fumonisins do not absorb ultraviolet light and do
not fiuoresce; therefore, derivatization reactions are required before detecting the
toxins with chromatographic techniques. In order to visualize fumonisins on TLC
plates, Gelderblom et al., (1988a) and Cawood et al. (1991) used 0.5% p-
anisaldehyde which was able to react with amino group of fumonisins for
generating reddish purple compounds when heated to 120°C. Retention factor
(Rf) values of F81 and F82 developed on normal phase silica TLC (Merck, Art.
5554) in chloroform:methanol:acetic acid (60:35:10) are 0.32 and 0.52,
respectively (Ackerman, 1991). By spraying with fluorescamine, Rottinghaus et

al.(1992) visualized F81 and F8; on reverse-phase C18 TLC plate developed in

methanol:4% aqueous KCI (3:2). They observed the toxins under longwave
ultraviolet light. F 81 and F B: appeared as bright yellow—green fluorescent bands
at Rf values of 0.5 and 0.1, respectively. The detection limit of this procedure
was 0.1 ppm in corn and its recoveries from spiked corn samples averaged more
than 80%. In general, TLC methods are qualitatively useful and simple but they
are not accurate for quantifying fumonisins.

More accurate and lower detection limits for fumonisin quantification can
be achieved using HPLC which can be performed via formation of a maleyl
derivative that absorbed at 250 nm (Gelderblom et al., 1988a). The maleyl

derivative procedure was initially developed by Siler and Gilchrist (1982) for

67
analysis Altemaria altemata f. sp. Lucopersici (ALL) toxin which had a chemical

structure similar to fumonisins. Briefly, the toxin is dissolved in 0.1 M sodium
carbonate solution pH 9.2, and then maleic anhydride crystals are slowly added
over a period of 5 minutes. At least a 10:1 molar ratio of maleic anhydride
crystals to toxin is used in this procedure to ensure complete derivatization. Prior
to HPLC analysis, the maleylation mixtures are adjusted to pH 6-7 by dilution with
the mobile phase, 0.05M KH2PO42methanol (3:7, pH 3.5), or by addition of
hydrochloric acid. This procedure is good for detecting fumonisins in corn
cultures with Fusan’um monilifonne , which generally have a high (> part per
thousand, ppt) level of the toxins. lt, however, does not work for detecting
fumonisins in naturally contaminated corn (Norred, 1993).

HPLC sensitivity can be increased by formation of fumonisin-fluorescent
derivatives with o—phthaldialdehyde (OPA)(Shephard et al., 1990). OPA
derivatization should be performed between 1 and 2 minutes prior to HPLC
analysis due to the CPA—derivative stability (Shephard et al., 1990). Detection
limits of this procedure are approximately 50 nglg for F81 and 100 nglg for F82.
Scott and Lawrence (1992) developed a liquid chromatographic method using 4- -
fluoro-7-nitrobenzofurazan (NBD-F) which yielded moderately stable derivatives
and had detection limits of 1 nglg of F81 and F82. However, derivatization with
NBD-F required many steps and was time consuming.

Gas chromatography-mass spectrophotometry (GC-MS) has also been
applied to detecting fumonisins in corn and feeds (Plattner et al 1990). In this

method, an extract of sample is hydrolyzed in 1N potassium hydroxide at 60°C for

68
1 hour followed by acidification with 0.5N HCl to form aminopentol backbones

which are then analyzed with GC-MS. GC-MS is highly selective and sensitive
although it requires relatively expensive equipment.

Regardless of sensitivity and selectivity, HPLC and GC-MS methods are
expensive, complex, and time consuming. As an alternative to these methods,
immunoassays have already been proven to be useful in screening for other

mycotoxins (Pestka, 1988).

Fumonisin antibodies

Previously, both mouse polyclonal and monoclonal antibodies against
fumonisin 81, 82 and 83 were produced in our laboratory by using cholera toxin
(CT) as carrier and adjuvant (Azcona-Olivera et al., 1992a,b). Concentrations of
fumonisin 81, 82 and 83in buffer solution required for 50% binding inhibition were
260, 300, and 650 nglml respectively for polyclonal antibodies with Cl-ELISA, and
630, 1800, and 2300 nglml, respectively for monoclonal antibodies with CD-
ELISA. Recently, antibodies against fumonisins have been produced by several
other investigators. Fukuda et al. (1994) generated murine monoclonal antibodies
against F81 by using ovalbumin and keyhole limpet hemacyanin (KLH) as a
protein carrier. When these monoclonal antibodies were used in a Cl-ELISA, the
amount of F81 required for 50% inhibition ranged from 65 to 255 nglml. F81-
rabbit polyclonal antibodies have also been produced using a F81-KLH conjugate
(Usleber et al., 1994). When these rabbit antibodies were used in CD-ELISA,

69
only 0.6 ng/ml'of F8, was required to inhibit 50% of F81 peroxidase conjugate

binding to the solid-phase-bound antibodies.

Rationale.

An HPLC procedure approved by AOAC and IUPAC after throughout
collaborative studies has been the primary method used to detect F81. This
method, however, requires laborious sample extraction, cleanup, and elusions,
before finally detection. Several ELISA approaches have also been developed to
date (Azcona-Olivera et al., 1992a,b; Pestka et al., 1994; Tejada-Simon, 1994;
Fukuda et al., 1994; Usleber et al., 1994). The ELlSAs are preferable to HPLC
because of their simplicity, ease of sample preparation, and use of stable
reagents (El-Nakib, et al, 1981). However, detection of F81 in food samples by
CD-ELISA technique using monoclonal antibodies produced by Azcona-Olivera
(1992b) provided much higher (30 fold) fumonisin estimates than GC-MS or
HPLC method (Pestka et al., 1994). The CD-ELISA yielded even higher(up to 400
fold) fumonisin estimates in Fusarium moniliforme cultures (T ejada-Simon, 1994)
than that for HPLC. One possible explanation for these observations is that the
F81 antibody generated by using cholera toxin (CT) as the carrier-adjuvant is not
completely specific to fumonisins and, therefore, may have reacted with other
compounds found in Fusarium moniliforme cultures and contaminated grains. To
overcome these problems, it is desirable to produce antibodies which have higher
affinity and specificity to the toxins. By using KLH as a protein carrier, higher

affinity and specificity rabbit polyclonal antibodies against F81 could be generated

70
(Usleber et al., 1994). In addition, by utilizing hybridoma technique, monoclonal

antibodies which have higher specificity and affinity could be generated.

Based on the early findings, l hypothesized that use of KLH as a protein
carrier, polyclonal approaches and hybridoma techniques could result in the
development of higher affinity F81-specific antibodies. Specifically, the purposes
of this study were to: (1) produce both polyclonal and monoclonal fumonisin
antibodies using KLH as a protein carrier; (2) compare the applicability of these
antibodies in ELISA; (3) apply the optimal ELISA techniques for detecting F81 in
Fusarium corn cultures, corn and corn products; and (4) to compare the optimized

ELISA to the existing reference HPLC method.

MATERIALS AND METHODS

Chemical and reagents

All organic solvents and inorganic chemical were of reagent grade or
better. Ovalbumin (OA) (chicken egg albumin grade III; fraction Vll), Tween 20,
2,2’-azinobis(3-ethylbenzthiazolinesulfonic acid)(A8TS), hydrogen peroxide,
horseradish peroxidase (HRP)(fraction VI), sodium borohydride, glutaraldehyde,
penicillin/streptomycin solution (pen/strep) (100,000 units/ml), sodium pymvate,
polyethylene glycol (MW 1450) (PEG), hypoxanthine, aminopterin, thymidine,
pristane, and dimethyl sulfoxide were purchased from Sigma Chemical Co. (St.
Louis, MO). Bovine serum albumin (BSA)(Albumin, bovine fraction V) was
obtained from Amresco (Solon, Ohio). Dulbecco’s Modified Eagle’s Medium
(DMEM), NCTC supplemental medium, and fetal bovine serum (F88) were
obtained from Gibco Laboratories (Grand Island, NY). Tissue culture plasticware
was purchased from Corning Laboratory Science Co. (Corning, NY). The
myeloma cell line P3INS1l1-Ag4-1 (NS 1) (ATCC TIB 18) was purchased from
the American Type Culture Collection (Rockville, MD). Macrophage conditioned
media (MCM) was prepared as described by Sugasawara et al. (1985). Sheep
polyclonal antibodies used in this study were developed jointly by our laboratory

and Neogen Corp. (Lansing, MI).

71

72
Conjugation of fumonisin B1 to protein

Fumonisin 81 (F81) was conjugated using glutaraldehyde to KLH (Pierce,
Rockford, IL) for use as immunogen and to ovalbumin (fraction Vll) for use as a
solid-phase antigen for indirect ELISAs by the method of Avrameas and Temynck
(1969). The conjugation reaction was carried out at 4°C in 0.01M phosphate-
buffered (pH 7.4) saline (PBS). F 81 was added to 1 mglml suspension of carrier
protein at a protein/toxin weight ratio of 5:1 for FBi-KLH, and of 3.411 for F 81-OA.
An equal volume of 2% (vol.lvol.) glutaraldehyde in water was then added
dropwise with constant stirring. After 1 hour, the reaction was stopped by adding
sodium borohydride to a final concentration of 10 mglml. One hour later, the
mixture was dialyzed for 72 hours (three changes) against 0.01M PBS pH 7.2.
Both KLH and OA conjugates were aliquoted in fractions of 1 mg (total protein),
lyophilized, and stored at -2o°c.

F81 was conjugated to horseradish peroxidase (F81-HRP) at an HRP/F81
weight ratio of 8:1 and used in the competitive direct ELISA by the periodate
method (Nakane and Kawaoi, 1974). Two ml of HRP solution (8 mglml distilled
water) was reacted with 400 pl of fresh 0.1M sodium periodate (0.21 9 I10 ml
distilled water) for 20 minutes at room temperature to activate the HRP. The
solution was then dialyzed twice against 3 liters of 1mM sodium acetate buffer pH
4.4 overnight at 4°C. Activated HRP was added into the F81 solution (2 mg F81
in 200 pl of 50% acetonitrile in water) and 20 pl of fresh 1M sodium carbonate
(1.06 gl10 ml distilled water) was added dropwise to the solution. The conjugation

reaction was carried out for 2 hours at room temperature and then 200 pl of

73
sodium borohydride (4 mglml distilled water) was added dropwise. After

incubation for 2 hours at 4°C, the conjugate was dialyzed twice against 6 liter
0.85% (wt/vol.) sodium chloride overnight, lyophilized in small portions, and

stored at -20°C.

Rabbit immunization

Three Female New Zealand rabbits (Charles River Laboratories,
Wilmington, MA) were initially given a ten site subcutaneous (sc) injection with
F81-KLH conjugate. This injection consisted of 1.0 ml of the conjugate (0.5 mg
F81-KLHIml) in a 1:1 ratio of saline and Freund’s “complete“ adjuvant (Difco
Laboratory, Detroit, Michigan). Two weeks later, the rabbits were boosted
subcutaneously with 0.5 ml (0.5 mglml) of the conjugate in saline and Freund’s
“incomplete” adjuvant (Difco Laboratory, Detroit, Michigan) in the ratio of
1 :1(vol.lvol.). Every other week after the second injection, rabbits were bled from
lateral marginal earn vein. Two weeks after the fifth bleeding, rabbits were given
the second boost as the first one and two weeks later, the animal were sacrificed
and their blood was collected. Serum was obtained after overnight incubation of
blood at 4°C and centrifugation at 1,000 x g for 15 min. Rabbit immunoglobulins
were purified by 33 percent saturation with ammonium sulfate (Hebert et al.,
1973). Serum titer and antibody specificity were then determined by ELISA as

described below.

74
Hybridoma production

Eight female BALB/c mice (6 to 8 weeks of age, Charles River
Laboratories, Wilmington, MA) were immunized by intraperitoneal (i.p.) and
subcutaneous (s.c.) routes. Mice were immunized three times with F81 -KLH
conjugates at two-week intervals. The first immunization consisted of 200 pl
(0.25 pg F81-KLHI pl) of conjugate in a 1:1 (vol.lvol.) ratio of PBS and Freund’s
complete adjuvant. For the second and the third injections, mice received 5-50
pg F81-KLH conjugate which was mixed with an equal volume of Freund’s
incomplete adjuvant for ip injection or with an equal volume of saline for sc
injection. Ten days after the second and the third immunizations, ether-
anesthetized mice were bled from the tail vein and the blood was collected with a
heparinized tube. The blood was incubated at 4°C overnight and then centrifuged
at 1000 x g for 15 minutes to obtain mouse plasma. Titer and antibody specificity
were then determined by both direct and indirect ELISAs. Mice producing F81-
specific and high titer antibody were chosen for fusion. At 4 days prior to the
fusion, the mice were injected intravenously (iv) in the lateral tail vein with 4 pg
of F81-KLH conjugate in saline.

Hybridoma production was performed by a modification of the procedure of
Galfre and Milstein (1981). Spleen cells (1 x 10") from an immunized mouse
were fused with NS-1 myeloma cells (1 x 107) using PEG. Fused cells were
resuspended in 100 ml of complete DMEM medium supplemented with 1% NCTC
(vol.lvol.), 10 mM sodium pyruvate, 100 units/ml of pen/strep solution, 20%

(vol.lvol.) FBS, and 20% (vol.lvol.) MCM, and then distributed into 875 wells of

75
96-well plates and incubated for 24 hours at 37°C in a humid atmosphere of 5%

CO2 in air. One half of the supernatant from each well was removed and
replaced with an equal volume of HAT medium (complete medium with
hypoxanthine, aminopterin, and thymidine). This operation was repeated every 3
days during a 2 week period, after which time HAT medium was eliminated
gradually and replaced by HT medium (the same composition of HAT but without
aminopterin). Supematants of hybridoma cultures were tested for the presence
of F81 specific antibody by Cl-ELISA. Cultures that produced a F81 specific
antibody were successively scaled up and cloned by limiting dilution at 0.5-1
cell/well (Goding, 1980). Subclones yielding desirable antibody activity were then
isolated and stored in FBS-dimethyl sulfoxide (9:1) under liquid nitrogen. Mass
production of F81 monoclonal antibodies was done by expansion of the selected
subclones. Antibodies were purified and concentrated from cell-free culture
supematants by precipitation with 50% saturated ammonium sulfate (Hariow and
Lane,1988)

For large scale production of monoclonal antibodies in vivo, mice were
injected with 0.5 ml of pristane (2,6,10,14 tetramethylpentadecanoic acid)
intraperitoneally (Potter, 1972). After 7-10 days, the mice were immunized with
5x105 to 5x10‘5 hybridoma cells from an actively growing culture which had been
centrifuged and resuspended in 0.5 ml 20% F BS-DMEM. After mouse-abdominal
swelling (usually 7-10 days after hybridoma injection), ascitic fluid was tapped
with an 18 gauge needle, and then purified by 45-50% saturation of ammonium

sulfate (Harlow and Lane, 1988).

76
Indirect ELISA

Indirect ELISA was performed by a modification of the procedure of
Azcona-Olivera et al. (1992a) and used to determine serum titers. Briefly, wells
of polystyrene microtiter plates (lmmunolon 4, Dynatech Laboratories, Alexandria,
VA) were coated overnight (at 4°C) with 100 pl of F81-OA (5 pglml) in 0.1 M
sodium carbonate-bicarbonate buffer (pH 9.6). Plates were washed six times by
filling each well with 300 pl of 0.02% (vlv) Tween 20 in PBS (PBS-Tween) and
aspirating the contents. Nonspecific binding was blocked by filling the wells with
300 pl of 1% (wt/vol.) bovine serum albumin in PBS 0.01 M (BSA-PBS), pH 7.2.
After incubating for 30 minutes at 37°C, the plate was washed six times with
PBS-Tween. Fifty pl of serially diluted mouse serum was added to each well and
incubated at 37°C for 1 hour. Wells of serially diluted preimmune serum were
used as controls. Unbound antibodies were removed by washing six times with
PBS-Tween, and 100 pl of goat anti-mouse lgG peroxidase conjugate (2 pglml, in
BSA-PBS, Cappel Laboratories, West Chester, PA) was added to each well. The
plate was incubated for 30 minutes at 37°C and washed eight times with PBS-
Tween. Bound peroxidase was determined with ABTS substrate (1 ml of 35 mg
ABTS/15 ml distilled water mixed with 11 ml of citrate buffer pH 4 and 8 pl
hydrogen peroxide) as described previously by Pestka et al. (1982). Absorbance
at 405 nm was read with a Vmax Kinetic Microplate Reader (Molecular Devices

Corporation, Menlo Park, CA). Titer of each serum was arbitrarily designed as

77
the maximum dilution that yielded twice or greater absorbence as the same

dilution nonimmune control serum.

A competitive indirect ELISA (Cl-ELISA) was used to verify specificity of
antibodies in sera toward F 81 during the course of immunization and to determine
those tissue culture wells which contained hybridomas secreting desirable
antibody, following fusion and cloning. Briefly, microtiter plates were coated and
blocked as described in the indirect ELISA procedure, and then 50 pl of standard
F8, (0 - 5000 nglml) dissolved in PBS was simultaneously incubated with 50 pl of
appropriate dilution of antibody or culture supernatant over the FBi-OA solid

phase for 1 hour at 37°C. The assay was then completed as described above.

Direct ELISA

Direct ELISA was used to determine the titer of animal sera (Azcona-
Olivera et al., 1992a). Plates were coated (125 pllwell) with serial dilution of
preimmune and immune sera in 0.1 M sodium carbonate-bicarbonate buffer
(coating buffer pH 9.6), then incubated overnight by drying at 40°C in a
conventional oven. After washing and blocking, 50 pl of F8. -HRP (2 pglml, in
BSA-PBS) was added to each well. After 1 hour of incubation at 37°C, plates
were washed and bound peroxidase was determined as described above.

A competitive direct ELISA (CD-ELISA) was used for F81 detection in
fungus cultures, corn and corn products. Briefly, microtiter plates with 96 wells
were coated and blocked as described in the direct ELISA, and then 50 pl of

serially (0 to 25 nglml) diluted F81 standard in 10% methanol or of appropriately

78
diluted samples in 10% methanol were simultaneously incubated with 50 pl of

HRP-F81 (2 pglml in 1% BSA-PBS) for one hour at 37°C. The assay was then

completed as described in the indirect ELISA.

Veratox®

Veratox® (Neogen Corp, Lansing, MI) is an ELISA kit for fumonisin test
based on CD-ELISA and was developed jointly between our laboratory and
Neogen Corp. Veratox® was used to detect F 81 in corn and corn products and
performed according to its instructions. Briefly, 100 pl of serially diluted F81
standard (0-20 pglml) or diluted samples was put into red-marked microtiter wells
and then simultaneously added with 100 pl of conjugate solution from the blue-
labeled bottle. This solution was mixed by pipetting the liquid up and down 3
times using an 8 or 12 -channel pipetor. One hundred pl of the liquid mixture
was transferred into coated-antibody micro titer wells and incubated for 15
minutes at room temperature. Every 30 seconds, the wells were swirled back
and forth carefully to mix the solution. After 15 minutes, the solution was
decanted. The wells were washed 5 times by filling/decanting with distilled water,

and then added 100 pl substrate solution from the green-labeled bottle to

develop color. The color development was then stopped by addition of 100 pl of
stop reagent from the red-labeled bottle. Absorbance at 650 nm was read with a
Vmax Kinetic Microplate Reader (Molecular Devices Corporation, Menlo Park,

CA).

79
ELISAGRAM

ELISAGRAM was used to determine F83 and other compounds in food
and culture samples which are also bound to FBi-antibodies. It was performed
as described by Pestka (1991) with modification. Briefly, fumonisin B1 in samples
was separated using silica gel 60 thin layer chromatography (Kieselgel 60, 0.063-
0.200 mm, Merck, 'SA) with chloroformzmethanolzacetic acid (60:30:10) as
developing solvents. Nitrocellulose membranes (0.45 pm; Schlicher and Schuell,
Keene, NH) were coated with F81 antibodies by soaking them in F81 antibody
solution over night. The TLC plate was sprayed with PBS and then blotted with
the FBi-antibody-coated nitrocellulose (NC) membranes. The AB-coated NC
membranes were then soaked in FB1-HRP solution (2pglml in 1% BSA-PBS) and
incubated at room temperature for 10 minutes. NC sheets were washed with
PBS containing 0.2% Tween 20, and then rinsed briefly in distilled water. For
color development, the NC sheets were incubated for 10-20 minutes at 25°C with
10 ml of dioctylsodium sulfosuccinate (DONS)I3,5,3’,5’-tetramethylbenzidine
(TMB) substrate prepared as described by Koch et al. (1985). The reaction was
stopped by washing the NC in distilled water and then incubating for 10 minutes
in stopping reagent solution (100 mg DONS dissolved in 13 ml of ethanol and
made up to 50 ml with distilled water). After drying between filter paper, the
white spots on the NC indicating the presence of F81 toxin in the samples were

observed.

80
Fusarium cultures

Fusarium cultures used in this study were M-5986 F. moniliforme, isolated
from samples that produced PPE (Ross et al., 1990); and M-5982 F. moniliforme,
isolated from samples that produced ELEM (Ross et al., 1990). In addition, F.
gramineamm W-8, which did not produce fumonisins, but rather deoxynivalenol
and zearalenone (Marasas et al., 1984a), was used as a negative control. These
cultures were obtained from the Fusarium Research Center at Pennsylvania
State University (University Park, PA).

Fusarium monilifomie cultures were inoculated onto petri dishes with V-8
juice agar medium (Stevens, 1974). Each liter of V-8 juice agar medium
contained 200 ml of V-8 juice, 3 g of calcium carbonate, and 20 g of agar
(Jackson and Bennet, 1990). These plates were incubated for 10-14 days at
room temperature on an alternating light-dark schedule. To prepare spore
suspensions, a loopfull of conidia from F. monilifomie petri dish cultures was
transferred to V-8 juice agar slant-tubes which were then incubated at room
temperature for 10-14 days on an alternating light-dark schedule. Spore
suspensions were obtained by washing the slant tubes with sterile distilled water.

Unlike Fusarium monilifonne spores, conidial suspensions of
F.graminearum W-8 were prepared by transferring the cultures into an ,150—ml
Erlenmeyer flask containing 40 ml of autoclaved CMC medium (15 g of
carboxymethyl cellulose, 1 g of ammonium nitrate, 1 g of potassium phosphate,
0.5 g of magnesium sulfate heptahydrate, 1 g of yeast extract in 1 liter of distilled

water) (Stevens, 1974). The flasks were then incubated at 25°C with shaking at

81
220 rpm. Cultures were checked between 3 and 5 days for macroconidia

production. Conidial suspensions were obtained by filtering CMC medium
through 4 layers of sterile cheesecloth to remove mycelia.

Prior to inoculation, 250-ml Erlenmeyer flasks containing 40 g of ground
corn and 11 ml of distilled water were autoclaved for 30 min. After autoclaving,
an additional 11 ml of sterile distilled water containing 107 conidia was added
(Stevens, 1974). The flask cultures were incubated in the dark at 25°C for 14 to
28 days. Corn cultures were extracted with 5 ml/g of acetonitrilezwater (1:1) by
soaking for 2-3 hours with mixing every half hour (Plattner et al., 1992).
Suspensions were filtered through Whatman # 1 filter paper (Whatman Ltd.,

Maidstone, UK) and the filtrate was tested for fumonisin 81 by ELISA and HPLC.

Com-sample extraction and clean up

Com extraction was performed by a modification of the procedure of Thiel
et al. (1993). Food, feed, or fresh corn samples were finely ground with Waring
Blender Model 1042 (VVinsted, Connecticut). A subsample (5 g) was put in 50
ml plastic-conical tubes, and 25 ml of 75% (vol.lvol.) methanol in distilled water .
was added. The tubes were then shaken (American Rotator V, R4140) at 200
rpm for 20 minutes. The solution was centrifuged at 10009 for 10 minutes, and
filtered through Whatman # 4 filter paper (Whatman Ltd., Maidstone, UK). The
filtrates were diluted with 10% methanol at ratio of 1:40 or higher before analyzing
for fumonisin 81 with CD-ELISA and Veratox® (Neogen Corp., Lansing, MI,

48912).

82
For some experiments, filtrates were cleaned up by passage through a

strong anion exchange (SAX) column (Bond-Elute SAX cartridges, 3 cc capacity
containing 500 mg sorbent; Varian, Harbor City, CA 90710) before analyzing with
HPLC. Briefly, pH of the filtered extract was checked and adjusted, if necessary,
with 0.1 M KOH to pH 5.8-6.5 before running through the SAX column. SAX
columns were attached to 16-port vacuum manifold (Alltech, State College, PA)
and conditioned by washing successively, first with 5 ml methanol and then with 5
ml of 75% (vol.lvol.) methanol in water at flow rate of no more than 2 mllmin.
While maintaining the flow rate, 10 ml of the substrate was applied to the column.
The column was then washed with 8 ml of 75% methanol (vol.lvol.) in water,
followed by 3 ml methanol.

Fumonisins were eluted with 10 ml of 1% (vol.lvol.) acetic acid in
methanol, at flow rate of no more than 1 mllminute (at atmospheric pressure).
The eluent was collected in 15 ml conical tubes. One half of that (5 ml) was
evaporated in 4 ml capacity vials under stream of nitrogen at about 60°C. The vial
was then capped and stored in 4°C until HPLC analysis. Prior to F81 detection

with HPLC, the purified sample residue in the 4 ml vial was redissoved with 100

pl methanol.

HPLC

Measurement of F 81 by HPLC was performed according to the previously
described reference method (Shephard et al., 1990 and Sydenham et al., 1992).

The liquid chromatography system used in this study consisted of lsco Model

83
2300 HPLC pump with an injector valve (Valco valve)(Lincoln, NE); H-S3 C13

#316 stainless steel packed 013 column (Perkin Elmer, Norwalk, Co) 0258-0178
reverse phase (3.30m); Hewlett Packard model HP 3392A integrator (Avondale,
PA); Linear Instruments Fluor fluorescence detector LG. 304 (Reno,NE) fitted
with a 3.1 pl flow cell, 500 psi (34 atm) maximum pressure and set at 334 nm
(excitation) and 440 nm (emission) and slit widths of 12 nm or similar. The
mobile phase was methanol:0.1M sodium dihydrogen phosphate (13.89
NaH2PO4.H2O in 1 liter distilled water)(66:34) that adjusted to pH 3.4 with ortho-
phosphoric acid and filtered through 0.22 pm water GV membrane (Milipore
Corporation, Bedford, MA). Before running the mobile phase, the C13 column
was washed approximately for 2 hours with degassed absolute methanol at a flow
rate of 1.0 mllminute and then with 25% (vol.lvol.) methanol in water for approx.
2 hours at the same flow rate. The mobile phase was pumped at 1.5 mllminute
when analyzing samples. After analyzing all samples, the column was washed
with 25% (vol.lvol.) methanol in water then with absolute methanol for
approximately two hours each at a flow rate of 1.0 mllminute.

OPA derivatizing reagent was prepared by dissolving 40 mg O-
phthaldialdehyde in 1 ml methanol and diluted with 5 ml 0.1 M sodium borate (3.8

g Na2B4O-, in 100 ml dH2O) and 50 pl of 2-mercaptoethanol. The solution was

stored no more than one week at room temperature in the dark.

Purified sample residues in a 4 ml vial were dissolved in 100 pl methanol.
Fumonisin standards were serially diluted (0.5 to 100 pglml) in methanol. Twenty

five pl fumonisin standard or samples were transferred to the base of small test

84
tubes and mixed with 225 pl OPA reagent. Less than 2 minutes after mixing, 10

pl of the mixture was injected into the HPLC system.

Statistics

Linear regression analyses were used to correlate ELISA and HPLC data.
A linear correlation coefficient (r) equaling to 1 indicates there is a perfect positive
relationship between the ELISA and HPLC methods, and rclose to -1 indicates a
strong negative correlation; whereas, a correlation coefficient close to 0 indicates
no relationship between two variables (Steel and Torrie, 1980). The P value is
the probability of being wrong in concluding that there is a true association
between the variable. The smaller the P value, the greater the probability that the
variables are correlated. Sigma Plot® (Scientific Graph System, Version 1.00 for
Windows, Jandel Scientific, San Rafael, CA) was used to perform linear

regression analyses.

RESULTS AND DISCUSSION

Rabbit polyclonal antibodies

Three rabbits were immunized and boosted with FB1-KLH immunogen.
Each rabbit was bled from a lateral marginal ear vein and its serum was prepared
and stored. Titers of the rabbit polyclonal antibodies in a direct ELISA format
were much lower (more than 3,200) as compared to that in an indirect ELISA
format (more than 64,000) (Figure 2.1). Concentrations of F81 required for a
50% inhibition of antibody binding were much lower (approximately 100 nglml) in
CD-ELISA than that in Cl-ELISA (more than 10,000 nglml) (Figure 2.2). In a
direct ELISA format, antibodies were bound to a solid phase and then enzyme-
labeled antigen (FB1-HRP) was added to detect and Quantify the antibodies.
Since F81 was conjugated to HRP directly without using glutaraldehyde as “a
bridge”, it is possible that FB1-HRP conjugate was reacted with only F 81 specific
antibodies. On the other hand, unlike in FB1-HRP conjugation, glutaraldehyde
was used as “a bridge” in both F81-OA and FBi-KLH conjugations. Therefore,
glutaraldehyde-0A might have acted as an epitope for glutaraldehyde specific
antibodies to produce non-specific binding as well as specific antibodies binding
to F81. If this happened, then both specific and non-specific binding between
rabbit antisera and the coating protein would be detected upon addition of the

second enzyme-labeled antibodies (goat-anti-rabbit lgG-HRP). In that case, an

85

86
indirect ELISA format would yield greater color development, and subsequently

higher titer than the direct ELISA. However, F81 required to inhibit 50% of the
antibody binding may be much more higher in Cl-ELISA as compared to that in

CD-ELISA.

Mouse immunization and monoclonal antibodies

Four mice were injected intraperitoneally and the other four were
immunized subcutaneously with FBi-KLH conjugates. All immunized mice
produced F81 specific antibodies as early as 4 weeks after initial exposure of F 81-
KLH immunogen. Competitive inhibition ELISAs for F81 using mouse antisera
were determined with both Cl- and CD-ELISA formats (Figures 2.3 and 2.4). Like
F81 rabbit antibodies binding, mouse antiserum binding was inhibited by F81 to a
greater extent in CD-ELISA than in Cl-ELISA. CD-ELISA results were used to
select optimal mice for fusion.

Mice were sacrificed and their spleen cells were fused with myeloma (NS1)
cells. The fused cells were then cultured into 875 wells of 96-well plates.
Hybridomas were detected in 821 wells, indicating a fusion efficiency [(number of
wells with growing colonies/number of wells seeded) x 100%] of 94 %. This
fusion efficiency was similar to that observed by Azcona-Olivera et al. (1992b).

After screening with CI-ELISA using F81-BSA coated plates, more than 20
wells were identified as FB1-antibody producers. Of these, seven cell lines
(A703, 8103, B1G5, 82F7, B406, B4E3, B4611) remained stable during scale-

up prior to cloning. Cl-ELISA standard curves of these cell lines are shown in

87
Figure 2.5. Concentrations of F81 required for a 50% inhibition of antibody

binding ranged from 90 to 2000 nglml. The two best cell lines (82F7, B4D6) were
cloned by limiting dilution twice and from these cultures, four cell lines (N205,
Q1C5, QZC9, R185) were chosen for large scale antibody production in ascitic
fluid. Mice were injected with activer growing hybridoma cells (N2C5, Q1C5,
0209, R185) after intraperitoneal injection with pristane. After mouse-abdominal
swelling, ascitic fluids of F81 -monoclonal antibodies were tapped and purified.
Titers and competitive inhibition using these antibodies were then analyzed with
both CD- and Cl-ELISA. Titers exceeded 40,000 with CI-ELISA and were
between 500 to 1,000 with CD-ELISA (Figure 2.6). Concentrations of F81
required for a 50% inhibition of antibody binding for the clones ranged from 50 to

100 nglml with CI-ELISA and from 400 to 800 nglml with CD-ELISA (Table 2.1).

Comparison among different antibodies

F81 Rabbit polyclonal and N2C5 monoclonal antibodies were compared to
the 205 monoclonal antibody which was previously generated by Azcona-Olivera
(1992b) using F 81 cholera immunogen and to sheep antisera developed against
FB1-KLH conjugate (Neogen Corp., Lansing, MI).

As previously noted, CD-ELISA for F81 using rabbit polyclonal antibodies
was more sensitive than CI-ELISA (Figure 2.2). However, the opposite was true
for the monoclonal antibodies (Figure 2.7). In order to ascertain which F81
antibodies had the highest avidity, CD-ELISA standard curves for F81 polyclonal

antisera and CI-ELISA standard curves for F81 monoclonal antibodies were

88
generated (Figure 2.10). Concentrations of F8, required for 50% inhibition of

antibody binding were 6, 80, 100, and 200 nglml for sheep, rabbit, N2C5, and
205, respectively (Table 2.2). Thus, the sheep antisera contained F81 specific
antibodies with the highest affinity. These results support the hypothesis of this
study [use of KLH as a protein carrier, polyclonal approaches and hybridoma
techniques could result in the development of higher affinity F81-specific
antibodies than the 205 antibodies which were previously produced by Azcona-
Olivera et al. (1992a,b) in our laboratory].

The affinity of F81 polyclonal antisera was higher than that of the
monoclonal antibodies (Table 2.2). This observation may be related to several
possibilities. Firstly, when producing monoclonal antibodies, hybridomas
secreting F81 antibodies with lower affinity, instead of with higher affinity, might
be chosen during hybridoma screening. Secondly, hybridomas secreting higher
affinity F81 antibodies might not have been stable and died before screening.
Thirdly, the affinity of F8. antibodies might be affected by animal species since
sheep, rabbits and mice produced F81 antibodies with different affinities. This
current study result agreed with the findings of Usleber et al. (1994) and Fukuda -
et al. (1994) who also generated F81 antibodies using KLH as a protein carrier.
The former researchers produced FB1-rabbit polyclonal antibodies that required
only 0.6 nglml F81 for 50% inhibition with CD-ELISA. Meanwhile, the latter
researchers generated F81 monoclonal antibodies that required a higher

concentration of F81 (65 nglml) for 50% inhibition with CI-ELISA.

89
Since the F81 sheep antisera contained FB1-specific antibodies with the

highest affinity, they were utilized in CD-ELISA for detection of F81
concentrations in fungal corn cultures, corn and corn products. CD-ELISA
standard curves for F81 , F82 and F83 using these sheep antisera were
graphically shown in Figure 2.11. Cross reactivities of the sheep antibodies
towards F81, F82, and F83 [determined as (nglml of F81 required for 50%
inhibition)l(ng/ml of fumonisin analogue required for 50% inhibition) x 100] were

100, 24 and 30%, respectively (Table 2.3).

Detection of FB1-like compounds

Several investigators reported that ELISA methods tended to give higher
F 81 estimates than HPLC methods. Minervini et al. (1992) found ELISA methods
yielded higher F81 estimates than HPLC when applied to Italian feed samples.
Elevated estimates for F81 in feeds by ELISA as compared to HPLC have also
been found in foods (Pestka et al., 1994) and in Fusarium corn cultures (Tejada-
Simon, 1994). The latter investigator reported that ELISA methods yielded much
higher F81 estimates (up to 400 times) than HPLC. She proposed that one
reason for the different F 81 estimates between the two analytical methods might
have resulted from the presence of compounds structurally similar to F81 in
sample extracts which were detectable by ELISA, but not by HPLC.

Detection of the F81-Iike compounds in corn and corn products might be
performed by ELISAGRAM. F81 monoclonal antibodies (205) produced by

Azcona-Olivera et al., (1992b) and F81 -sheep antisera developed jointly by our

90
laboratory and Neogen Corp. (Lansing, MI) were used in the ELISAGRAM.

Before analyzing samples, a standard of F81 (2.5 pglml) was used to test
whether the ELISAGRAM system worked or not for F81 toxin. Twenty pl of the
F81 standard was directly spotted on silica gel thin layer chromatography, and
then transferred onto FB1-antibody-coated nitrocellulose membrane (NC). The
NC was incubated with F 81 -HRP, washed and finally incubated in dioctylsodium
sulfosuccinate (DONS)I3,5,3’,5’-tetramethylbenzidine (T MB) substrate for color
development. However, a white dot on the NC did not appear. This indicated
that F81 standard did not bind to a coating antibody, so the coating antibody
reacted with FB1-HRP which subsequently produced green color after incubation
in HRP-substrate solution. Similar observations occurred when 10 pg F81
standard was directly spotted onto the NC. Thus, the ELISAGRAM that worked
well for zearalenones and aflatoxins (Pestka, 1991) did not work at all for F81
toxin. Since F81 easily dissolved in water, the toxin might have run off during
washing or blotting before reacting with coating F81 specific antibodies on the
NC. As a result, a white dot on the NC was not developed although a relatively
high quantity of F81 standard (10 pg) was spotted on the NC. Thus the

ELISAGRAM would not be useful for detecting putative F 81 -like compounds.

Detection of F81 in Fusarium cultures
F81 analysis was performed by HPLC and CD-ELISA using F81 -sheep
antisera (Neogen Corp., Lansing, MI). F. graminearum (FW-8), F. proliferatum

(M-5956), and F. moniliforme (M-5958) were grown in solid (ground corn) media.

91
Non FB1-producing F. graminearum (FW-8) and uninoculated samples were

used as negative controls. The corn cultures were harvested at 2, 3, 4, and 5
weeks, and then extracted with 50% acetonitrile, filtered, and subsequently
analyzed with HPLC and CD-ELISA.

Standard curves for CD-ELISA and for HPLC analyses were generated
and shown graphically in Figures 2.10 and 2.12, respectively. Based on these
standard curves, F 81 concentrations in Fusarium corn cultures were calculated
and the results were tabulated in Table 2.4. F 81 concentrations in the cultures
ranged from 0 to 220 pglg with HPLC and from 0 to 759 pglg with ELISA.

In both negative controls, F 81 was not detected by either HPLC or ELISA.
However, when this ELISA was used to assay F81 in fumonisins producing
cultures, it yielded F81 estimates higher (mean = 2.8-fold) than HPLC method
(Table 2.4). These current study results were a vast improvement over the
findings of Tejada-Simon (1994) who found that ELISA methods yielded much
higher F81 estimates (up to 400-fold) than HPLC. The improvement was likely
caused by the sheep antiserum which had higher affinity and specificity than 205

monoclonal antibodies used by Tejada-Simon (1994) (Table 2.2).

Detection of F8. in corn and corn product

Corn and corn products analyzed in this study consisted of 77 fresh com,
14 com food and 28 com feed samples. Ground corn samples were extracted
with 75% (vol.lvol.) methanol in distilled water. After filtering through Whatman

#4 filter paper (Whatman Ltd., Maidstone, UK), the raw methanolic extracts were

92
diluted at a 1:35 and then analyzed by CD-ELISA using FB1- sheep antisera

(Neogen Corp, Lansing, MI). Although detection of F81 in the food and feed
extracts at a dilution of 1:35 with the CD—ELISA could be performed successfully,
a problem was found when analyzing F 81 in the fresh corn extracts. After color
development, some samples had a higher color intensity (higher 0.0.) than the
zero F81 standard solution. CD-ELISA standard curves for F81 in extractant
[10% methanol (vol.lvol.) in distilled water], and raw methanolic extracts of fresh
corn sample numbers 37 and 56 (at a dilution of 1:35) were graphically shown in
Figure 2.8. All standard curves had relatively the same pattern, but different 00.
indicating that the raw corn extracts contained an interfering material which was
able to generate non specific binding. Several possibilities might relate to this
observation. First, the corn extracts might contain peroxidase which then reacted
with peroxidase substrate (ABTS) resulting increase of color intensity. Second,
the corn extracts might contain protein compounds which could bind to both
sheep antisera and FBi-HRP resulting higher color intensity alter the addition of
ABTS. Third, the pH of the corn substrate might favor to the reaction of sheep
antisera with F81 -HRP or with peroxidase-containing compounds which
subsequently increased color intensity. Fourth, the corn extracts might contain
non protein compounds which could increase color intensity.

Several efforts have been performed to solve the above problem. The
corn extracts was heated and then centrifuged to inactivate peroxidase and to
eliminate protein compounds from the corn extracts. Addition of NaCI to the corn

extracts was also performed to precipitate the interfered proteins. Measurements

93
of corn substrate pH and adjustment of the pH to neutral were also carried out to

eliminate the effect of the sample pH. All of these efforts, however, did not
solved the problem indicating that the interfered compounds were not peroxidase
or proteins. The problem was eliminated after the corn extracts were diluted up
to 200 times or higher with 10% methanol in distilled water. By diluting sample
extracts, Laamanen and Veijalainen (1992) were also able to eliminate a similar
problem when they analyzed T-2 toxin in milled grains with an ELISA method.
After diluting fresh corn extracts up to 200 times, CD—ELISA standard curves for _
F8. in the extractant and in the diluted corn extracts had relatively the same
0.0. and pattern (Figure 2.9).

Beside the CD-ELISA, Veratox® (Neogen Corp, Lansing, MI) and HPLC
were also used to detect F81 in fresh com, com products and spiked-com
samples. After sample extraction and filtration, raw methanolic extracts were
analyzed for F81 using both CD-ELISA and Veratox® methods. Some raw
extracts were cleaned up with SAX columns and then analyzed by HPLC. A
standard curve of HPLC (Figure 2.13) and standard curves of CD-ELISA and
Veratox® (Figure 2.10) for analyzing F81 in corn and corn products were then
developed. Based on these standard curves, F81 concentrations in com-spiked
samples, corn, and corn products were calculated.

Alter calculating F81 concentrations in com-spiked samples containing
F81 of 100 to 3000 nglg, the F81 concentrations in the samples were divided by
the added F 81 concentrations and then multiplied by 100% to determine percent

recovery of the mycotoxin (Table 2.5). Percent recoveries of F81 in SAX-

94
columned samples with HPLC ranged from 68.8 to 84.7 percent (average = 74.1)

(Table 2.5) indicating some F81 might loss during extraction and SAX-column
purification. These recovery results were relatively similar to the findings of Stack
and Eppley (1992) who analyzed F81 in SAX-columned extracts of com-spiked
samples containing F81 of 500 to .2000 nglg with HPLC, and found percent
recoveries of F 81 in the range of 63.2 and 71.5 percent. Meanwhile, by using the
same method Usleber et al. (1994) reported that percent recoveries of the same
toxin in corn samples spiked with 50 nglg and 500 nglg F81 were 63.2% and
65.6%, respectively.

When both Veratox® and CD-ELISA were used to analyze F81 in the
com-spiked samples, percent recoveries of F 81 in the raw methanolic extracts
were higher than that in SAX-cleaned extracts (Table 2.5), indicating that some
F81 might have lost during SAX column purification. F81 could not be detected in
SAX-clean extracts from the samples which were spiked with only 100 or 200
nglg F81 (Table 2.5) because all of the spiked toxin might have lost during
purification. Percent recoveries of the spiked F81 in the raw methanolic corn
extracts determined with CD-ELISA and Veratox® ranged from 48.5 to 127.2%
(Table 2.5). This variability might result from two possibilities. Firstly, the
presence of F81 in the corn extracts might not be distributed evenly during
spiking, so some samples yielded high recoveries and the other ones provided
low recoveries. Secondly, the F81 -extraction efficiencies from corn containing

different amounts of F81 might differ. Because of high variability, these percent

95
recoveries were not used to adjust the concentration of F81 in corn and corn

products.

F81 concentrations in 14 food samples ranged from <0.04 to 7.11 pglg
(part per million, ppm), 0.13 to 9.59 ppm and <0.02 to 10.16 ppm when detected
by HPLC, Veratox® , and CD-ELISA, respectively (Table 2.6). The ratio of
Veratox® and CD-ELISA results to HPLC results ranged from 1.0 to 1.5 (Table
2.6). These ratio results were significant improvement over the findings of Pestka
et al. (1994) who reported that the ELISA estimates were higher (up to 30-fold)
than HPLC estimates. As in Fusarium corn cultures, sheep antiserum used in
this currentstudy had greater affinity and higher sensitivity than 205 monoclonal
antibodies used by Pestka et al. (1994) (Table 2.2). These undoubted
contributed to the analytical improvement. These results indicate that both
Veratox® and CD-ELISA were suitable to routinely screen com-based foods from
F81.

The levels of F81 in Italian feed samples ranged from <0.04 to 4.99 ppm,
0.34 to 7.63 ppm and 0.27 to 8.43 ppm when measured by HPLC, Veratox®, and
CD-ELISA, respectively (Table 2.7). Veratox® and CD-ELISA analyses yielded ‘
F 81 estimates approximately twice higher (mean = 2.24 and 1.93 respectively)
than HPLC method. Regression analyses between HPLC and Veratox® and
between HPLC and CD-ELISA had linear correlation coefficients (r) of 0.961 and
0.951, respectively (Table 2.7) indicating that HPLC had a strong positive
relationship with both Veratox® and CD-ELISA methods. These current study

results were much better than the finding of Minervini et al. (1992) who reported

96
that the correlation coefficient between HPLC and ELISA was only 0.240,

indicating no relationship at all between ELISA and HPLC methods. Like the
above mentioned assays of Fusarium cultures and foods, the sheep antiserum
was critical to ELISA improvement.

Seventy seven of fresh corn samples were first analyzed by Veratox® to
screen for samples contaminated with F81. All positive samples and
approximately half of the negative samples were reanalyzed by Veratox®, C0-
ELISA and HPLC. Analysis of positive fresh corn samples, Veratox® and CD-
ELISA yielded higher F81 estimates (mean = 2.85 and 1.54, respectively) (Table
2.8) than HPLC. However, the three methods yielded the same results when
applied to negative fresh corn samples (Table 2.8). Linear correlation coefficients
(r) between HPLC and Veratox® and between HPLC and CD-ELISA were 0.933
and 0.836, respectively (Table 2.8) indicating that HPLC correlated well with both
Veratox® and CD-ELISA methods. Thus, both Veratox® and CD-ELISA can be
useful for routinely screening fresh corn from F81 .

CD-ELISA developed in this current study was much better than the
previous CD-ELISA that was developed by Azcona-Olivera et al. (1992a,b) and
used by Minervini et al. (1992), Pestka et al. (1994) and Tejada-Simon (1994) for
analyses of F 81 in feeds, foods, and fungal cultures, respectively. However, when
this “new" CD-ELISA was used to detect F81 in corn cultures, corn foods, corn
feeds and fresh corn, it still provided higher F81 levels (means = 2.8, 1.3, 1.9,
and 1.5, respectively) as compared to those obtained by HPLC (Tables 2.4, 2.6,

2.7, 2.8). This disagreement might relate to several possibilities. Firstly, HPLC

97
detected only F81 ; whereas, CD-ELISA detected not only F81 but also F82 and

F 83 since F81 sheep antisera used in the CD-ELISA cross-reacted with F82 and
F83 (Table 2.3). Secondly, the sample extracts might contain F81 -similar
compounds which were detectable by CD-ELISA but not by HPLC. The F81 -
sheep antisera might not be completely specific for F81, so they can possibly
detect other related compounds. Thirdly, because it contains an amine (-NH2)
functional group, F81 might react with protein compounds prior to or during
extraction to form F 81 -protein conjugates that cross react with F81 -sheep
antisera, and thus increase the response of the CD-ELISA without affecting

HPLC detection.

CONCLUSION

Mouse monoclonal antibodies, rabbit and sheep polyclonal antibodies against
F81 were readily generated after immunization with keyhole limpet hemacyanin
(KLH) as a protein tamer. The sheep antisera had the highest affinity and were
most specific to F 81 as compared to the mouse monoclonal and rabbit polyclonal
antibodies when they used in competitive inhibition ELISA Cross reactivities of
these sheep antisera toward fumonisin B1 , 82 , and 83 were 100, 24 and 30%,
respectively.

C03ELISA using these F81 sheep antisera provided approximately two
fold higher F B. estimates than HPLC when used for detection of F 81 in Fusarium
corn cultures, corn and corn products. This was much better than CD-ELISA
previously developed in our laboratory In addition, this CD-ELISA as well as
Veratox® correlated well with HPLC methods, suggesting that it is suitable for

routinely screening Fusarium cultures, corn and corn products from F 83 toxin.

98

99

 

0.0. (405 NM)

’4
,5“
if”
/x)
// ,////’D
// o’ // A
// / // /
(I -O-— /U/ //
// — ' RFi // / //
/ // __D—- R24 / // K/
V // —-A—-- R3,; / / /
/// / / /
// / / /
///// “0‘ R110 / / /
./// V 324’ / / /
A/// —<>— R,-p / / /
I

 

 

 

 

Figure 2.1.

 

ANTIBODY DILUTION

ELISA titration of rabbit polyclonal F81 antibodies. Sera were
obtained two weeks after the second injection with FB1-KLH
immunogen. Each data point represents the average value of
duplicate measurements. Filled symbols indicate indirect ELISA.
Open symbols are direct ELISA. R1, R2 and R3 refer to rabbit
number. The letter i indicates immunized and p indicates preimmun.

100

90

80

70-1

PERCENT INHIBITION

30—l

20—

101

Figure 2.2.

100

 

60—

50

IIT ITWI IIII TTTI TTIW

T

 

Ijfl ITIT

IITI

 

 

TIITTIII

 

l l l
100 1000 10000

PB, (nglml)

Competitive inhibition ELISA for F81 using different rabbit polyclonal
antibodies. Sera were obtained two weeks after the second injection
with FBi-KLH immunogen. Each data point represents the average
value of duplicate measurements. Filled symbols indicate Cl-ELISA.
Open symbols are CD-ELISA. R1, R2 and R3 refer to rabbit number.

101

 

100

BO—I

704

8
I

IIrI TllI‘rIIrIITI

PERCENT INHIBITION
8
1

1o—

 

IITj

IIII

VIII

IPWTIIII

IIII

 

 

 

 

 

F8, (nglml)

Figure 2.3. Competitive inhibition ELISA for F81 using mouse sera. Sera were

obtained ten days after the third subcutaneous (SC) injection with
FB1-KLH immunogen. Each data point represents the average
value of duplicate measurements. Filled symbols indicate Cl-ELISA.
Open symbols are CD-ELISA. Subscript 1-4 refers to mouse
number.

102

 

100

IIII

IIII

00—

TTTI

N
c
I

ITIVIIII

PERCENT INHIBITION
S
l

Ir—II—ITITI

IIII

10“

 

ITII

ITII

 

 

 

 

111 l 1 111111 I 141L111

 

I l
100 1000 10000

FE, (nglml)

Figure 2.4. Competitive inhibition ELISA for F81 using mouse sera. Sera were

obtained ten days after the third intraperitoneal (IP) injection with
FB1-KLH immunogen. Each data point represents the average
value of duplicate measurements. Filled symbols indicate Cl-ELISA.
Open symbols are CD-ELISA. Subscript 1-4 refers to mouse
number.

103

 

110

IIrI

too—

IIII

I—I—TI

00-4

st
0
l

PERCENT INHIBITION

 

IIII IIII IIII IfiIITIIII IITI ITIT ITrI IIII

 

 

'10 111111 1 1 111111 J 1 111111 1 1 111111

10 100 1000 10000
FB1 (nglml)

 

Figure 2.5. Competitive indirect ELISA curves for F81 using hybridoma
supematants. Supematants were obtained from hybridoma cells
producing F 81 antibodies before cloning. Each data point represents
the mean value of duplicate measurements.

104

 

10-—

T I T I

2.5 —l

FT If

ZD'~

I I I I

1A5—

o.o. (405 NM)

I I I j

L0-—

I I I I

05-—

I T I j

00-—

_I

N2C5

R185

 

 

 

 

10‘

Figure 2.6.

10‘ 1o4 104
ANTIBODY DILUTION

ELISA titration of monoclonal F81 antibodies prepared from ascites
fluid. Each data point represents the mean value of duplicate
measurements. Filled symbols indicate Cl-ELISA. Open symbols are
CD-ELISA. Q1C5 clone had no titer when determined with CD-
ELISA.

105

 

 

100 t
E
so .1
: . N2C5
so—: A R185
: v
70 __ o1cs
: I ozcs
2 3°“-
0 _
E _
m _-
5 5° -
g l-
i— I
E 4o—
0
E
Q 0

 

N
a 8

I I
rWIIfiTWI ITIj

 

 

IITI rIII

 

.10 1111 1 1 L11111 1 1 111111 1 111L111 1 1 111111

1 10 100 1000 10000
FB1 (nglml)

Figure 2.7. Competitive inhibition ELISA for F81 using monoclonal antibodies
prepared from ascitic fluid. Data points represent the mean value of
duplicate measurements. Filled symbols were Cl-ELISA. Open
symbols were CD-ELISA. Q1CS clone had no titer when determined
with CD-ELISA.

106

 

 

 

 

 

 

 

Table 2.1. Sensitivity of Cl-and CD-ELISAs for F81 using monoclonal antibodies
prepared from ascites fluid
Amount of F 81 required for 50% inhibitiona (nglml)

Clone Cl-ELISA CD-ELISA

N2C5 50 400

R1 85 60 700

Q1C5 90 -"

QZC9 100 800

 

 

 

a Data from Figure 2.7

b No titer in this assay.

 

107

 

 
   
     

 

 

     

 

1.2 —
I.
~ v CORN EXTRACT#56(1:35)
1.0 I A CORN EXTRACTII 37 (1:35)
- 0 EXTRACTANT
0.0 —
g _
no J”
8 0.0
RI :
o
0.4 —
0.2 a
o.o___jJ11I 1 _1 111111 1 1 111111
0.0 0.1 1.0 10.0 100.0
F31 ("9"”)

Figure 2.8. Competitive direct ELISA curves for F81 using F81 sheep anti sera
(Neogen Corp., Lansing, MI). F8, was dissolved in extractant [10%
methanol (vol.lvol.) in distilled water], fresh corn extract numbers 37
and 56 at a dilution of 1:35). Each data point represents the mean
value of triplicate measurements.

108

 

1.2 -

L.
r v CORN EXTRACT If 50 (1:200)
.
1.0 —-h A CORN EXTRACT 11 37 (1:200)
- 0 EXTRACTANT
0.0 —

/

0.6 -i ‘\

0.0. (405 NM)

T I I I

0.4 —

0.2 I \

001111 4 1111111 1 1 111111 1 1111111 1 1111_11
I

0.0 0.1 1 .0 10.0 100.0
FB1 (nglml)

IITW

I r I I

 

 

Figure 2.9. Competitive direct ELISA curves for F81 using F81 sheep anti sera
(Neogen Corp, Lansing, MI). F81 was dissolved in extractant [10%
methanol (vol.lvol.) in distilled water], fresh corn extract numbers 37
and 56 at a dilution of 1:200). Each data point represents the mean
value of triplicate measurements.

109

 

110

100 -

90—

80

PERCENT INHIBITION
'5’ 8 3 S S 3‘
1 1 1 1 1

.5
O
L

a
I

IITT

O VERATOX

ITII

C SHEEP

IIIW

A R:

IIIV

V 2D5 MAB

 

IIII
CI

R1 BS MAB

 

 

 

IIII ITII IIII ITTI TIII IIII TTFT
.\
.

 

 

1 1 1 11111 J1 1 L1111L 111 1 11111 1 1 111111 1 1 111111 1 1 1 11111

 

I
d
O
O
0

I I I I
10.0 1 00.0 1 000.0 1 0000.0

FB, (nglml)

Q
P
.5
.L
C

Figure 2.10. Comparison of competitive inhibition ELISA for F8, mouse-

monoclonal antibodies, rabbit-and sheep-polyclonal antibodies, and
Veratox®. Monoclonal antibodies (N205 MAB and 205 MAB) were ‘
prepared from ascitic fluids. N205 MAB was generated in the current
study using F81-KLH immunogen, while 205 was produced by
Azcona-Olivera (1992a) using F81-0holera toxin immunogen. R3
refers to rabbit number 3. Sheep antiserum was from a single lot
supplied by Neogen Corp. (Lansing, MI). Veratox" is an ELISA kit for
F81 based on CD-ELISA and is produced and supplied by Neogen
Corp. (Lansing, MI). Monoclonal antibodies were determined with Cl-
ELISA, but polyclonal antibodies were measured with CD-ELISA.
Each data point represents the mean :1: standard errors of the mean
(11 = 4, two of duplicate measurements). Absorbencies at 0 nglml F 81
ranged from 0.675 to 1.250.

110

Table 2.2. Comparison of competitive F 81 ELISA using mouse-monoclonal
antibodies prepared from ascites fluid, rabbit-and sheep-polyclonal

 

 

 

 

 

 

 

antibodies
F81 concentration Absorbance value
. required for 50% for 0 ng F 81 lml

Antibody Inhibition“ (nglml) ELISA format
205b 200 1.10 Cl-ELISA
N205° 100 1 .25 Cl-ELISA
R3° 80 0.79 CD-ELISA
Sheep" 6 0.74 CD-ELISA
Veratox' 6 0.68 CD-ELISA

 

 

 

 

" Data from Figure 2.8.

b Monoclonal antibodies produced by Azcona-Olivera (19928) using F 81-0holera
toxin immunogen.

° Monoclonal antibodies generated in the current study using F B1-KLH
immunogen .

d Rabbit polyclonal antibodies from rabbit number 3.
e Sheep polyclonal antibodies from Neogen Corp. (Lansing, MI).

' Veratox is an ELISA kit for F81 based on CD—ELISA and is produced and
supplied by Neogen Corp. (Lansing, MI).

 

111

 

100

IIII

90—1

ITII

80— 0 F32

IIrI

70—

VIII

004

504

IIIrTrfirI

PERCENT INHIBITION

30-1

ITIIfiITfi

20-

10«

TPIIHIII

 

 

o 1 111111 1 _1 J_1_1111 1 1 11111—11

 

1 10 100
F8, (nglml)

Figure 2.11. Competitive direct ELISA standard curves for F81 , F82 and F83 ,
using F81 sheep antisera. Data for sheep antisera were provided by
Dr. Mohamed M.Abouzied (Neogen Corp., Lansing, MI). Each data
point represents the average value of triplicate determination.

112

Table 2. 3. Cross reactivity of F81 sheep antisera toward fumonisin analogues.

 

 

 

 

 

 

 

Fumonisin Fumonisin concentration required Cross reactivity (%)°
analogue for 50% inhibition8 (nglml)

F 81 5.5 100

FB2 22.9 24

F83 18.3 30

 

a Data from Figure 2.9.

b Cross reactivity defined as (nglml of F81 required for 50% inhibition)l(nglml of
fumonisin analogue required for 50% inhibition) x 100.

 

113

 

 

 

 

 

600
. v - 5.384x + 2.224
50° "I E - 0.999
400 —
g 300 —
.- _
5 r-
2 200 -—
L.
100 _
1.
o .—
r-
1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 r 1 1 l 1
0 20 40 00 00 100 120

F81 (uglmi)

Figure 2.12. A typical “broad range “ standard curve used for HPLC analyses of
Fusarium corn cultures. Each data point represents the average
value of two determinations.

114

 

1000

"' Y I 01.789 X + 3.991
800 —

I" 3 0.998

600 —

400 -—

AREA 0: 1000)

200 -

 

 

1111 41111111 1111 11114114 111L

 

 

0 2 4 6 8 10 12
F8, (nglml)

Figure 2.13. A typical “low range “ standard curve used for HPLC analyses of
corn and corn products. Each data point represents the average
value of two determinations

115

Table 2.4. Comparison of F81 concentrations in Fusarium corn cultures
detected by HPLC and CD-ELISA methods

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SAMPLE HPLC (pglg) CD—ELISA (pg/g)“ RATIO OF ELISA TO HPLC
Controlj’, wk. 2 <0.04° <0.02h -
Control, wk. 3 <0.04 <0.02 -
Control, wk. 4 <0.04 <0.02 -
Control, wk. 5 <0.04 <0.02 -
FW-B c, wk. 2 <0.04 <0.02 -
FW-8, wk. 3 <0.04 <0.02 -
FW-8, wk. 4 <0.04 <0.02 -
FW—8, wk. 5 <0.04 <0.02 -
101-5955 ‘1, wk. 2 7.0 19.3 2.8
M-5956, wk. 3 33.9 30.3 0.9
M-5956, wk. 4 9.7 26.1 2.7
M-5956, wk. 5 21.4 39.7 1.9
M-5958 °, wk. 2, R1 23.8 58.1 2.4
M-5958, wk. 3, R1 9.8 27.4 2.8
M-5958, wk. 4, R1 17.9 58.1 3.2
M-5958, wk. 5, R1 24.3 76.2 3.1
M-5958, wk. 2, R2 8.9 24.2 2.7
M-5958, wk. 3, R2 220.0 759.0 3.5
M-5958, wk. 4, R2 <0.04 <0.02 <0.02-
M-5958, wk. 5, R2 25.7 109.0 4.2
MEAN :I: SEM r 2.8 :1 0.9

 

' CD-ELISA using F81 sheep antisera (Neogen Corp., Lansing, MI); Linear regression analyses
between HPLC (X) and CD-ELISA (Y) at P<0.05 yielded an equation line of Y = 3.428X - 7.640
and linear correlation coefficient (r )=0.992.

b Uninoculated samples.

" F. proliferatum cultures (M-5956).
' Standard errors Of the means.

" CD-ELISA detection limit (20 nglg)

° Fusarium graminearum cultures(FW-B).
° Fusarium moniliforme cultures (M-5958).
9 HPLC detection limit (0.04 nglg)

wk. 2, 3, 4, and 5 refer to week number 2, 3, 4, and 5 respectively at which Fusarium cultures
were harvested.

R1 and R2 refer to replication number 1 and 2, respectively.

116

Table 2.5. Comparison of F81 recoveries from spiked-com samples containing
F81 of 100 to 3,000 pglg using raw methanolic extracts and SAX-
Cleaned extracts as determined by HPLC, Veratox® ', and CD—ELISA

using F 8. sheep antisera (Neogen Corp., Lansing, MI)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PERCENT RECOVERIES " of F8. in
F8. RAW METHANOLIC EXTRACTS SAX-CLEANED EXTRACTS
ADDED
nglg Veratox® CD-ELISA HPLC Veratox® CD-ELISA
100 86.3 3 24.0 nd 68.8 3 4.2 nd nd
200 127.2 3 31.8 48.5 3 3.3 71.2 3 12.4 nd nd
600 80.5 3 12.4 70.3 3 13.3 84.7 3 7.2 56.5 3 6.4 50.2 3 9.1
1,000 71.5 3 6.8 70.0 3 2.3 73.6 3 9.9 50.2 3 10.7 58.7 3 9.6
3,000 60.5 3 6.4 50.1 3 4.5 72.1 3 5.4 51.6 3 9.1 38.8 3 1.7
MEAN° 85.2 3 16.3 59.7 3 5.9 74.1 3 7.8 45.2 3 16.3 49.2 3 6.8

 

a Veratox® Fumonisin Test, Neogen Corp.(Lansing, Ml).

" (F81 found/F81 added) x 100%.

° Calculated from positive recoveries.

nd indicates no F81 detected (Detection limits of Veratox® and CD-ELISA were 0.02 pglg) ;
Thus, recovery could not be calculated.

Each data point represents the mean 3: SEM (n = 3).

SEM = standard errors of the means.

 

117

Table 2.6. Comparisons Of F81 concentrations in 14 com-food samples by
HPLC. Veratox® ‘, and CD-ELISA using F81 sheep antisera
(Neogen Corp., Lansing, MI) and the ratios of Veratox®a and CD-
ELISA to HPLC results

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SAMPLE FUMONISIN B1 (p919) FOUND” BY RATIOS TO HPLC °
NUMBER “ HPLC Veratox®' CD-ELISA Veratox®” CD-ELISA

- 41 <0.04' 0.13 0.02' - -
38 <0.04 0.15 0.29 - -
43 <0.04 0.17 0.12 - -
48 <0.04 0.14 0.07 - -
33 0.34 0.44 0.41 1.3 1.2
32 0.42 0.58 0.59 1.4 1.4
25 0.64 0.94 0.85 1.5 1.3
10 0.75 0.96 1.16 1.3 1.5
8 0.92 0.97 1.28 1.1 1.4
7 1.10 1.24 1.08 1.1 1.0
22 1.21 1.61 1.20 1.3 1.0
6 1.79 2.35 2.25 1.3 1.3
21 2.27 3.21 2.40 1.4 1.1
H9 7.11 9.59 10.16 1.3 1.4

MEAN 1.18 1.61 1.56 1.31 1.26

smo 1.84 2.47 2.59 0.12 0.20

 

 

 

 

 

 

 

' Veratox® Fumonisin Test, Neogen Corp. (Lansing, MI).

b Linear regression analyses between HPLC (X) and Veratox® (Y1) and between HPLC and CD-
ELISA (Y2 ) at P < 0.05 yielded equation lines of Y, = 1.473X - 0.485; r= 0.992 and Y2 =
1.370X - 0.356; r= 0.983.

c Veratox® or CD-ELISA results divided by HPLC results.

‘ Ranked according to HPLC results; the sample descriptions were on Appendix (Table 5.1).
‘ HPLC detection limit (40 nglg).

' CD-ELISA detection limit (20 nglg).

’ Standard errors of the means.

118

Table 2.7. Comparisons of F 81 concentrations in 28 Italian feed samples by
HPLC, Veratox® ‘, and CD-ELISA using F81 sheep antisera
(Neogen Corp., Lansing, MI) and the ratios of Veratox®a and CD-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ELISA to HPLC results
SAMPLE FUMONISIN B1 (pglg) FOUND” BY THE RATIOS OF HPLC‘
NUMBERd HPLC Veratox®' CD-ELISA Veratox®“ CD-ELISA

F18 <0.04' 0.34 0.51 - -
F20 <0.04 0.49 0.27 - -
F8 <0.04 0.51 0.87 - —
F25 0.24 0.62 0.41 2.6 1.7
F3 0.11 0.40 0.38 3.6 3.4
F22 0.21 0.43 0.15 2.1 0.7
F1 0.22 0.95 0.61 4.3 2.7
F16 0.24 0.56 0.68 2.3 2.8
F11 0.34 0.90 0.74 2.6 2.2
F24 0.36 0.72 0.88 2.0 2.5
F23 0.38 1.25 0.74 3.3 2.0
F30 0.42 0.67 0.32 1.6 0.8
F13 0.44 1.01 0.58 2.3 1.3
F10 0.50 1.07 0.74 2.2 1.5
F29 0.62 1.12 0.79 1.8 1.3
F4 0.67 2.23 1.87 3.3 2.8
F7 0.79 1.62 2.85 2.0 3.6
F28 0.94 1.16 1.44 1.2 1.5
F9 0.94 3.05 2.30 3.2 2.4
F2 1.00 1.69 0.88 1.7 0.9
F14 1.07 2.06 1.73 1.9 1.6
F27 1.16 2.37 1.66 2.0 1.4
F19 1.24 2.40 1.93 1.9 1.6

 

 

119

Table 2.7. (Con’d)

 

 

 

 

 

 

 

 

 

SAMPLE FUMONISIN B1 (pg/g) FOUND” BY THE RATIOS OF HPLC‘
NUMBER“ HPLC Veratox®“ CD-ELISA Veratox®“ CD-ELISA
F17 1.31 2.37 2.60 1.8 2.0
.33, 1.39 2.17 1.96 1.6 1.4
F26 1.67 2.96 3.84 1.8 2.3
F15 3.71 5.01 8.43 1.3 2.3
F12 4.99 7.63 7.87 1.5 1.6
MEANI 1.00 1.86 1.86 2.24 1.93
SEMO 1.11 1.60 2.10 0.77 0.77

 

 

 

 

 

 

 

 

'Veratox® Fumonisin Test, Neogen Corp. (Lansing, MI).

b Linear regression analyses between HPLC (X) and Veratox ® (Y1) and between HPLC (X) and
CD-ELISA (Y2 ) at P < 0.05 yielded equation lines of Y. = 1.010X + 0.532; r= 0.961 and
Y2 =1.300X + 0.160; r= 0.951).

°Veratox® or CD-ELISA results divided by HPLC results.

° Ranked according to HPLC results, and the sample descriptions were on Appendix (T able 5.2).
° HPLC detection limit (0.04 nglg).

' Mean of positive samples according to HPLC results.

9 Standard errors Of the means

120

Table 2.8. Comparisons of F 81 concentrations in fresh com 3 samples by
HPLC, Veratox® 3, and CD-ELISA using F 81 sheep antisera
(Neogen Corp., Lansing, MI) and the ratios of Veratox®b and CD-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ELISA to HPLC results
SAMPLE FUMONISIN B. (pglg) FOUND6 BY THE RATIOS OF HPLC‘I
NUMBER“ HPLC Veratox®” CD-ELISA Veratox®” CD-ELISA

P14 <0.04' <0.02“ <0.02 " - -
P15 <0.04 <0.02 <0.02 - -
P19 <0.04 <0.02 <0.02 - -
P21 <0.04 <0.02 <0.02 - -
P25 <0.04 <0.02 <0.02 - -
P27 <0.04 <0.02 <0.02 - -
P28 <0.04 <0.02 <0.02 - -
P31 <0.04 <0.02 <0.02 - -
P32 <0.04 <0.02 <0.02 - -
P37 <0.04 <0.02 <0.02 - -
P46 <0.04 <0.02 <0.02 - -
P5 <0.04 <0.02 <0.02 - -
P50 <0.04 <0.02 <0.02 - -
P51 <0.04 <0.02 <0.02 - -
P52 <0.04 <0.02 <0.02 - -
P53 <0.04 <0.02 <0.02 - -
P54 <0.04 <0.02 <0.02 - -
P55 <0.04 <0.02 <0.02 - -
P56 <0.04 <0.02 <0.02 - —
P58 <0.04 <0.02 <0.02 - -
P59 <0.04 <0.02 <0.02 - -
P62 <0.04 <0.02 <0.02 - -
P67 <0.04 <0.02 <0.02 - -
P7 <0.04 <0.02 <0.02 - -
P74 <0.04 <0.02 <0.02 - -
P9 <0.04 <0.02 <0.02 - -
P17 <0.04 <0.02 <0.02 - -
P26 <0.04 <0.02 <0.02 - -
P63 <0.04 <0.02 <0.02 - -
P42 0.04 0.27 0.26 7.1 6.8
P61 0.05 0.26 0.16 5.2 3.1
P80 0.05 0.25 <0.02 4.6 0.0

 

 

121

Table 2.8. (Con’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SAMPLE FUMONISIN B, (11919) FOUND6 BY THE RATIOS OF HPLC“
NUMBER' HPLC Veratox®° CD—ELISA Veratox®° CD-ELISA
P10 0.08 0.16 <0.02h 2.0 0.0
P12 0.10 0.28 <0.02 2.8 0.0
P57 0.10 0.23 <0.02 2.2 0.0
P8 0.12 0.21 <0.02 1.7 0.0
P1 0.15 0.31 0.15 2.0 1.0
P66 0.25 0.75 0.60 3.0 2.4
P29 0.28 0.52 0.34 1.9 1.2
P45 030 0.47 0.86 1.5 2.8
P20 0.47 0.76 0.88 1.6 1.9
P44 0.70 0.92 0.56 1.3 0.8
MEANi 0.20 0.41 0.29 2.85 1.54
SEMl 0.21 0.23 0.33 1.75 1.95

 

 

 

 

 

' Harvested in five counties of Michigan in Summer 1994. Samples were supplied by Dr. Patrick
L. Hart (105 Pesticide Research Center, Michigan State University, Ml, 48824). All samples
were first analyzed with CD-ELISA; and then all positive samples as well as randomly selected
negative samples were analyzed with HPLC, Veratox®, and CD-ELISA. .

” Veratox® Fumonisin Test, Neogen Corp. (Lansing, MI).

° Linear regression analyses between HPLC (X) and Veratox® (Y3) and between HPLC (X) and
CD-ELISA (Y2 ) at P < 0.05 yielded equation lines of Y1 = 1.127X + 0.039; r= 0.933 and
Y2 = 0.973X + 0.008; r = 0.836.

dVeratox® or CD-ELISA results divided by HPLC results.
° Ranked according to HPLC results, and the sample descriptions were on Appendix (Table 5.3).

F 9- " Detection limit of HPLC (40 nglg), Veratox® (20 nglg), CD-ELISA (20 nglg), respectively.
' Mean Of positive samples.

jStandard errors Of the means.

122

PART III

PRODUCTION OF DEOXYNIVALENOL (VOMITOXIN) ANTIBODIES

ABSTRACT

PRODUCTION OF DEOXYNIVALENOL (VOMITOXIN) ANTIBODIES
By

Sutikno

Deoxynivalenol (DON) was reacted with hemisuccinate (HS) for 8 hours to
form DON-HS derivatives. These derivatives were conjugated to bovine serum
albumin (BSA) and then used as an immunogen for producing DON specific
antibodies. Twenty female BALBIC mice were immunized intrasplenically,
intraperitoneal, or subcutaneously and 6 female New Zealand rabbits were injected
intramusculariy with the immunogen. All animals apparently produced high titer
antisera. However, these antisera could not be used in competitive ELISAs for DON
and also did not cross react with either 3-acetyl-DON, 15-acetyl-DON, nivalenol,

4,15—diacetylnivalenol or fusarenone—X.

123

INTRODUCTION

Natural occurrence.

Deoxynivalenol (DON, vomitoxin) is a trichothecene mycotoxin, produced
by Fusan'um graminearum and Chemically determined to be 301, 7a, 15-trihydroxy-
12, 13-epoxytrichotheC-9-en-8-one (Table 3.1)(Yoshizawa and Morooka, 1973).
DON was first isolated in Japan from Fusarium-infected barley (Morooka et al.,
1972). This toxin was also found in the United States by Vesonder et al. (1973)
who analyzed Northwest Ohio corn infected with Fusan‘um. These investigators
tested the presence of the emetic compound by intubation of each fraction of their
isolation procedures into pigs to induce vomiting at which they isolated and
Characterized DON as an active compound. Because of its emetic effect on
swine, DON was also named vomitoxin (Vesonder et al., 1973). As a
consequence of these findings, surveys for the presence of DON in foods and
feeds as well as testing for its toxicological and immunosuppresive effects began.

Surveys for the presence of DON in foods especially grain and grain
products have been done by several investigators in several countries and the
results are summarized in Table 1.3. DON has been found worldwide and is a
major contaminant in cereal grains (Tanaka et al., 1988; Hietaniemi and

Kumpulainen, 1991). DON production occurs when environmental conditions in

124

125

Table 3.1. Structures of deoxynivalenol (DON) analogs (Adapted from Ueno,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 983)
Compound R1 R2 R3 R4 R5
DON OH H OH OH =O
DON-3-HS OHS' H OH OH =O
DON-15-HS OH H OHS' OH =0
3—Acetyl-DON OAc" H OH OH =0
15-Acetyl-DON OH H OAc" OH =0
Nivalenol 0H 0H 0H 0H =0
4,15—Diacetylnivalenol OH OAo" OAob OH =0
Fusarenon-X 0H OAC" 0H 0H =0
‘ T-2 toxin 0H 0aCb Oacb H 0Ip

 

OHS', OAc", Olp° indicate 000(0H2)2000H, 0000H3, and 000H2CH(0H3)2, respectively.

 

126
the field are low temperature and high humidity (Cote et al., 1984; Vesonder et

al.,1978; Mills, 1982), although this toxin has also been found in high temperature
regions (Richardson et al., 1985). Vesonder et al. (1978) reported that cold and
wet weather tends to delay harvest and subsequently molds on the crop grow
continuously, and thus produce high quantities of DON. In some cases, however,
prolonged delay of harvest can yield a decline in DON concentration in grains left
in the field, possibly due to reaction with plant components or metabolism by
plant! fungal enzymes (Scott et al., 1984). In North America, the areas most
often contaminated by DON are the midwestem region of the United states

(Vesonder, 1983) and the eastern region of Canada (Seaman, 1982).

Toxicity

DON is much less toxic than other trichothecene mycotoxins such as T-2
toxin, HT-2 toxin, diacetoxyscirpenol, nivalenol, and fusarenon-X (Scott et al.,
1980). DON was found to be 60-fold less toxic than T-2 toxin and two fold less
toxic than nivalenol when the trichothecene toxicity is measured using lethal dose
50 (L053, an amount of toxin required to kill 50% population) of brine shrimp
larvae (Scott et al., 1980). In mice, the L050 of DON is 49 mglkg body weight
(intraperitoneal) and 78 mglkg body weight (oral) (Forsell et al., 1987). The
toxicity of this toxin may be due to protein synthesis inhibition (Ueno, 1983).

Consumption of high quantities of DON (at L053, or higher) results in
Classical acute symptoms of trichothecene toxicity ranging from necrosis of the

intestinal tract, bone marrow and lymphoid tissues as well as lesions in kidney

127
and heart, to death (Forsell et al., 1987; and Robbana-Bamat et al., 1987).

Pathological Changes include lesions and degeneration of the stomach and small
intestine mucosa, enlargement and edema of mesenteric lymph nodes, vascular
congestion and depletion of all lymphoid organs and liver (Cote et al., 1985;
Robbana-Bamat et al., 1987). At lower doses (<L050), ingestion of DON can
cause feed refusal, reduced growth rate, reduced milk production, reproductive
problems and cause immune alteration (Vesonder et al., 1973;1979; Cote et al.,
1984; 1985; Morrissey and Vesonder,1985; Whitlow and Hagler, 1987). The
toxicological effects of DON are gender-related with male or castrated male
animals being more sensitive to DON than female animals (Cote et al., 1985;
Greene et al., 1994).

In order to elucidate the immunotoxiCity of DON on animal, Pestka et al.
(1987) fed mice with DON-containing diets and then challenged the mice with
Listeria monocytogenes. They found that mouse resistance to the pathogen
decreased as indicated by the lower splenic Clearance of Listeria. Increased
splenic Listen’a counts following exposure to DON may be partly due to the toxin-
induced feed refusal rather than directly as a consequence of immune function
alteration of the animals (Pestka and Bondy, 1990).

Oral exposure of DON induces a dose dependent leukopenic effect in mice
in which white blood cells, lymphocyte and monocyte numbers are decreased
(Forsell et al., 1986; Robbana-Bamet et al., 1988) but neutrophil cells are
increased (Forsell et al., 1986). Increase Of an lgA-secreting cell number and

decrease of an lgG-secreting cell number in the Payer’s patch (PP) were also

128
reported in mice fed with DON (Pestka et al., 1990). In addition, ingestion Of

DON can also cause glomerular lgA deposition (Pestka et al., 1989) and a
dysregulation of serum immunoglobulin production by decreasing lgG and lgM
and increasing lgA and lgE serum levels (Forsell et al., 1986; Dong et al., 1991;
Pestka and Dong, 1993; 1994).

The presence of DON in both feed and food supplies is of concern as this
toxin occurs worIdwide (Table 1.3) and consumption of corn and mixed feeds
contaminated with DON have been associated with animal health problems.
Furthermore, food preparation and processing such as heating at 350°C,
Cleaning, milling and baking can not eliminate DON from food products (Young et
al., 1984). Reduced DON concentration alter food preparation results solely from
the dilution effect with clean wheat (Young et al., 1984). Thus, a combination of
worldwide occurrence of DON in grain and grain products and its resistance to
processing enable this mycotoxin to persist in the food and feed supplies.

In order to protect human and animal health, DON regulation has been
established in Canada and Russian Federation. The official DON tolerance limit
is 2.0 ppm in uncleaned sofl wheat in Canada and 0.5-1.0 ppm for foods in USSR
(Hietaniemi and Kumpulainen, 1991). However, DON has not been regulated yet
in the United States even though DON can be present in up to 50% of cereal and
cereal products tested (Abouzied et al., 1991). In the United States, the only
limitation of DON concentration in foods is an “advisory tolerance limit”
suggesting that wheat should contain not more than 2.0 ppm and finished

products for human consumption, should contain no more than 1.0 ppm

129
(Hietaniemi and Kumpulainen, 1991). The advisory levels for DON in wheat and

wheat products has been revised by the US. Food and Drug Administration
during 1993-1994 (Trucksess, 1995). The new levels are: (1) 1 ppm for brand,
flour, and germ for human consumption; (2) 10 ppm for grain and grain by-
products destined for ruminating beef cattle and feedlot cattle older than 4
months and for Chickens, with the added recommendation that the maximum level
of these ingredients in cattle or chicken diet is 50%; and (3) 5 ppm in grains and
grain by-products destined for pigs, and all other animals, with the added
recommendation that the maximum levels of these ingredients are 20% in the pig

diet and 40% in all other animal diets.

DON detection

Detection of DON in foods and feeds can be achieved with conventional
detection methods such as TLC, G0, HPLC and MS or with immunoassay
methods such as RIA and ELISA (Pestka et al., 1994a). These detection
methods have been reviewed in Part I. Notably, a monoclonal antibody against
. DON was generated by Casale et al. (1988) who derivatized DON to 3-0-
hemisuccinyl-DON after blocking two of the three available hydroxyl moieties with
a cyclic boronate ester. The detection limit of both direct and indirect ELISAs
employing the DON monoclonal antibodies in buffer solution was 200 nglml
(Casale et al., 1988). When these antibodies were employed in indirect ELISAs,
detection limit for DON in grain-based foods after dilution was 1000 ppb

(Abouzied et al., 1991). Later, higher affinity polyclonal antibodies against DON

130
were reported by Usleber et al. (1991) who derivatized DON to hemisuccinyl-

DON (HS-DON) without the blocking reaction. When the DON polyclonal
antibodies were used in the direct ELISA, it could detect DON in buffer solution
as low as 1.0 nglml (Usleber et al., 1991). This suggested that either the use Of
rabbits or the type of immunogen used by Usleber’s group resulted in an antibody

with improved affinity.

Rationale

Although conventional analytical methods such as TLC, G0, HPLC and
MS are very sensitive and selective, they generally require extensive and time-
consuming sample Clean up prior to DON detection (Pestka et al., 1994a). As an
alternative, ELISAS, which require simpler, quicker and easier sample
preparation, have also been developed (Casale et al., 1988; Usleber et al., 1991;
Abouzied et al., 1991). ELISAS using DON monoclonal antibodies, which were
developed in our laboratory, had high (1000 ppb) detection limit when applied to
food samples (Abouzied et al., 1991). Much lower detection limits Of ELISA
approaches using polyclonal antibodies have been demonstrated by Usleber et '
al. (1991). They suggested that reacting DON directly (without blocking reaction)
with succinic anhydride prior to coupling with protein carriers resulted in higher
affinity antibodies against DON.

In their attempts to produce DON antibodies, Zhang et al. (1986), Casale
et al. (1988) and Usleber et al. (1994) immunized animals either intraperitoneally,

subcutaneously or intradermally. Recently, intrasplenic immunization has been

131
proposed as a highly efficient technique for producing antibodies against small

amount of matrix bound antigen (Nilsson et al., 1987). This procedure is
applicable to the production of antibodies directed against highly conserved or
weakly immunogenic determinants (Van Ness et al., 1984). Thus, the
intrasplenic immunization technique may be capable Of eliminating the difficulties
of producing DON antibodies (Zhang et al., 1986; Casale et al., 1988, Usleber et
al.,1994).

Based on the above findings, I hypothesized that the use of DON-HS-BSA
conjugates prepared with the procedure of Usleber et al. (1991) for immunizing
either mice or rabbits via either intrasplenic, subcutaneous, intraperitoneal, or
intraderrnal injections would result in high affinity and specificity antibodies
against DON. The objectives of this study were to produce polyclonal or
monoclonal DON antibodies which have high affinity and specificity via
intrasplenic, subcutaneous, intraperitoneal, and intraderrnal immunizations with
DON derivative-BSA conjugation as described by Usleber et al. (1991), and then

compare the applicability of these antibodies in ELISA.

 

 

 

MATERIALS AND METHODS

Chemical and reagents

All organic solvents and inorganic Chemicals were of reagent grade or
better. Ovalbumin (OA) (Chicken egg albumin grade III; fraction VII), Tween 20,
2,2’-azinobis(3-ethylbenzthiazolinesulfonic acid)(A8TS), hydrogen peroxide,
horseradish peroxidase (HRP)(fraction VI), N-hydroxysuccinimide and 1,3-
dicyclohexyl-carbodiimide were purchased from Sigma Chemical CO. (St. Louis,
MO). Bovine serum albumin (BSA)(Albumin, bovine fraction V) was obtained from
Amresco (Solon, Ohio). Goat anti-mouse immunoglobulin G (lgG)-horseradish
peroxidase conjugate and goat anti-rabbit lgG—horseradish peroxidase conjugate
were Obtained from Cappel Laboratories (West Chester, PA). DON, 3-acetyl-
DON, 15—acetyl-DON, nivalenol, and fusarenone-X were purchased from Romer
Laboratories, Inc. (Washington, MO). Methofane (methoxy‘fiurane, inhalation

anesthetic for veterinary) was purchased from Pitman-Moore lnC. (Mundelein, IL).

Derivation Of DON

DON was derivatized by reacting with succinic anhydride according to the
procedures of Usleber et al. (1994) with slight modification. Briefly, DON (10 mg)
was dissolved in 1.0 ml of pyridine and then succinic anhydride (200 mg) was added.

This mixture was slowly stirred with a magnetic stirrer and heated at 100°C in a

132

133
steam bath for 8 hours. Pyridine was removed under N2 pressure and the residue

was dissolved in CHCI3 (15 ml). This solution was extracted with 0.1 M HCl (3x10
ml). The organic phase was dried under vacuum in a nitrogen flushing evaporator
and the remaining substance was re—dissolved in 2 ml of methanol. Results of the
derivatization reaction were detected with TLC methods using silica gel TLC plates
(Linear K High Performance Silica Gel # 4805-411, Whatman lntemational Ltd.,
Maidstone, England) developed in a solvent system of ethylacetatel n-hexanelacetic
acid at ratio of 75 : 25 : 5 (vol.lvol.lvol.). Unreacted DON and its derivative (HS-
DON) were visualized by spraying the developed TLC plates with 15% Al0l3 (15 g of
AICl3 dissolved in 85% alcohol in distilled water), heating at 110°C in a conventional
oven for 10 minutes, cooling in room temperature, and then observing the toxin
spots under 336 nm W light (Kamimura et al., 1981). The toxin and its derivatives
were also visualized by spraying the TLC plates with 1% (vol.lvol.) of 4(p—
nitrobenzyl)-pyridine in a mixture solution of chloroforrnzcarbon tetrachloride at a ratio
Of 2:3 (vol.lvol.), heating at 150°C, cooling at room temperature, spraying again with
10% (vol.lvol.) of tetraethylene pentamine in the mixture solution and then Observing
the toxin spot under 336 nm UV light (T akitani et al., 1979). The toxins appeared as
blue fluorescent spots. Alter a reaction of 8 hours, all DON was converted to HS-
DON which was then quantified with TLC methods, dried under nitrogen at room

temperature and stored in 4°C until conjugation reaction.

134
Conjugation of DON derivatives to protein

The HS-DON was coupled to protein [to bovine serum albumin (BSA) for use
as immunogen, to ovalbumin (OA) (fraction VII) for use as a solid-phase antigen in
indirect ELISA, and to horseradish peroxidase (HRP) for use as a labeled toxin in
direct ELISA] at a DON:protein ratio of 2:5 (w/w) by using an activated ester method
(Kitagawa et al., 1981). Briefly, HS-DON (5.0 mg) was dissolved in 0.5 ml N,N-
dimethyl fonnamide (DMF) and slowly stirred with a magnetic stirrer. Fifty pl of a
mixture containing 70 mg of N-hydroxysuccinimide and 60 mg of 1,3-dicyclohexyl-
carbodiimide dissolved in 1 ml DMF was added drop by drop and the mixture was
continuously stirred at room temperature for 30 minutes. Meanwhile, protein (12.5
mg) was dissolved in 1 ml sodium bicarbonate buffer (0.821 g of sodium bicarbonate
buffer dissolved in 100 ml distilled water), pH 7.2. The HS-DON solution was added
slowly to the protein solution and stirred for 2 hours at 4°C. This mixture was
dialyzed overnight three times against 4 liters of 0.01M phosphate buffer solution

(PBS) pH 7.2, aliquoted into 2 mg protein/tube, lyophilized and stored at -80°C.

Mouse immunization

Twelve female BALBIC mice (68 week old, Charles River, Laboratories,
Wilmington, MA) were injected subcutaneously (SC) and intraperitoneally (IP) with
DON-HS-BSA conjugates at two week intervals up to five times. The first
immunization was given at a dose of 50 and 100 pg conjugate/animal. In this
immunization, “complete “ Freund’s adjuvant (Difco Laboratory, Detroit, Michigan)

was used to emulsify the conjugate at a ratio (conjugateszadjuvant) of 1:1 (vol.lvol.).

 

 

135
The subsequent (booster) injections were at a dose of 25 and 50 pg of the

conjugate! mouse. For intraperitoneal injection, the conjugates were emulsified with
an equal volume of “incomplete” Freund’s adjuvant (Difco Laboratory, Detroit,
Michigan) and for subcutaneous injection, the conjugates were mixed with an equal
volume Of 85% of saline solution (85 g of NaCl dissolved in 100 ml of distilled water).
Ten days after every booster injection, methoxyflurane—anesthetized mice were bled
from their tail vein and the blood was collected within a heparinized tube. The blood
was incubated at 4°C over night and centrifuged at 1000 x g for 15 minutes to obtain
mouse plasma. Serum titer and serum specificity were then determined by indirect
ELISA methods.

In addition to the above, 8 female BALBIC (6-8 week old, Charles River,
Laboratories, Wilmington, MA) were given an intrasplenic immunization at a dose Of
20 pg/mice by the procedure of Nilsson et al. (1987). Briefly, mice were
anaesthetized using methoxyflurane before surgery. The animal was placed on its
right side and its lelt side fur was shaved. Alter swabbing the left abdominal with
70% ethanol, a cutaneous incision, about 10 mm long, was cut in the left
midscapular line followed by incision of the abdominal wall and peritoneum. The
caudal end of the spleen was carefully exposed, lying on fat and pancreatic tissue.
After a sterile nitrocellulose strip (1 x 2 mm) containing 20 pg of DON-HS-BSA was
deposited into the spleen by using a 20 gauge needle, the spleen was returned to
the abdominal cavity. The abdominal wall and the skin were sutured separately with
sterile thread. Alter two and four weeks, the mice were given the second and

third immunizations by IP and 30 injections at a dose of 10 and 50 pg Of the I

 

 

136
same conjugate/mouse, respectively. Ten days after each injection, the mice

were bled and the serum titers as well as specificity were determined by indirect

ELISA.

Rabbit immunization

Three female New Zealand rabbits (Charles River Laboratories,
Wilmington, MA) were given a ten site subcutaneous (SC) injection with 500
pglrabbit DON-HS-BSA conjugate which has been emulsified with Freund’s
“complete” adjuvant (Difco Laboratory, Detroit, Michigan) at a ratio of 1:1
(vol.lvol.). Four and ten weeks later, the rabbits were boosted with the same
immunogen but at half the dose. Freund’s “complete” adjuvant was replaced with
Freund’s “incomplete” adjuvant (Difco Laboratory, Detroit, Michigan) for boosting.
Another three female New Zealand rabbits were given a two site intramuscular
injection with 100 pg Of BSA-HS-DON conjugate per rabbit using Hunter's Titer
MaxTM (Cthx® Corporation, Norcross, GA) as an adjuvant. Four weeks later, a
booster injection was performed exactly the same as the first injection. Every
other week after the booster injection, rabbits were bled from lateral marginal ear
vein. Serum was obtained after overnight incubation of the blood at 4°C and
centrifugation at 1,0009 for 15 minutes. Rabbit immunoglobulins were purified by
ammonium sulfate precipitation according to the procedure of Hebert et al. (1973)
prior to determination of serum titer and antibody specificity with direct ELISA

techniques.

 

'3

rm-m

1 37
ELISA

Both direct and indirect ELISA were similarly performed as described in
Materials and Methods of Part II with the exception of toxin-0A conjugate, toxin-H RP
conjugate and blocking solution. In DON ELISAs, DON-HS-OA conjugate, instead
Of F81 -OA conjugate, was used for coating microtiter wells in indirect ELISA; DON-
HS-HRP conjugate, instead of F81 -HRP conjugate, was used as a labeled
mycotoxin in direct ELISA; and 1% (wt/vol) Of ovalbumin, instead of 1% (wt/vol) BSA,
in PBS 0.01 M, pH 7.2 was used to block non specific binding in both direct and
indirect ELISAs.

The titer of each senirn was arbitrarily Chosen as the maximum dilution that
yielded twice or greater absorbance as the same dilution of nonimmune control
serum. Each serum was tested for cross-reactivity with 3-acetyI-00N, 15-acetyl-

DON, nivalenol, 4, 15-diacetylnivalenol, and fusarenone-X.

RESULTS

DON derivatization

To introduce a carboxyl group into DON molecules, DON was reacted with
succinic anhydride. The end point of DON derivatization was monitored by TLC
methods after 2, 4, 6, and 8 hours of derivatization reaction. DON and its
derivatives were visualized with AICI3 and 4(p-nitrobenzyl)-pyridine reagents, and
both compounds were positive (appeared as a blue fluorescence spot) on the
TLC plates. This indicated that the derivative compound still possessed both
keto and epoxy moieties because AICI3 is specific for trichothecenes having 7-
hydroxy-8-keto side-chain residues (Kamimura et al., 1981) and 4(p—nitrobenzyl)-
pyridine was specific to compounds wntaining an epoxy group (Takitani et al.,
1979). The DON derivative ran as a single spot on the silica gel TLC plates
developed in a solvent system of ethylacetatel n-hexanelacetic acid at ratio of 75 :
25 : 5 (vol.lvol.lvol.). Retention fraction (Rf) values for DON and its derivative (HS-
DON) were 0.67 and 0.33, respectively. After heating for 8 hours, almost all DON
was already converted to HS-DON as indicated by a single spot on TLC plates at a

Rf value of 0.33.

138

139
Mouse immunization

Twelve mice were intraperitoneally (IP) and subcutaneously (SC)
immunized with BSA-HS—DON conjugate at a dose Of 50 and 100 pg/mouse. The
mice were then given four booster injections with the same conjugate. Ten days
alter each booster injection, the mice were bled from their tail vein and the serum
titer and specificity were determined by indirect ELISA. All mice produced
apparent high titers of antibodies (Figure 3.1) but their binding to solid-phase
bound DON could not be inhibited by as much as 20pglml of DON. These
antibodies also did not cross-react with either DON, 3—acetyI-DON, 15-acetyl-
DON, nivalenol, 4, 15-diacetylnivalenol, or fusarenone-X.

Another 8 mice were immunized with 20 p9 of BSA-HS-DON conjugate per
mouse via intrasplenic deposition. Two and four weeks later, the mice were
given the second and third immunizations by IP and SC injections at a dose of 10
and 50 pg of the same conjugate/mouse, respectively. Ten days after each
injection, the mice were bled and the serum titers as well as specificity were
determined by indirect ELISA. All of the animals could also produce relatively
high titer antibodies (Figure 3.2). However, when these antibodies were used in
indirect ELISAs, they could not detect DON although at high concentration (up to
20pglml). In addition, these antibodies were neither cross-reactive with '3-acetyl-

DON, 15-acetyI-DON, nivalenol, 4, 15-diacetylnivalenol, nor with fusarenone—X.

140
Rabbit immunization

Three rabbits were given a two site intramuscular injection with 100p9 of
BSA-HS-DON conjugate/animal using Hunter’s Titer Max” (Hunter’s adjuvant).
Another three rabbits were given a ten site subcutaneous injected with BSA-HS-
DON immunogen using Freund’s adjuvant. The animals were then given two
booster injections. Every two weeks after each booster, the rabbits were bled
and the antibodies were analyzed by direct ELISA to determine their titers and
specificities. All rabbits produced antibodies, but use of Freund’s adjuvant
resulted in higher titer antibodies than that of Hunter’s adjuvant (Figure 3.3).
Similar to mouse antisera, these rabbit antisera could not recognize free DON.
They were also not cross reactive with 3-acetyl-DON, 15-acetyl-DON, nivalenol,

4,15-diacetylnivalenol or fusarenone-X.

.n ‘T‘H_13“.il."_ _. .u
.

 

 

DISCUSSION

DON derivatization

The results of DON derivatization were similar to that of Usleber et al.
(1994) who also reported that all DON was already converted to its derivatives
after a reaction of 8 hours with succinic anhydride. Casale et al. (1988) have
analyzed the toxin derivatives by TLC and FAB-MS methods. They did not detect
DON, a Rf value of 0.84, but found at least three compounds (Rf values of 0.33,
0.60, and 0.68) when the derivative compounds were spotted on silica gel TLC
plates developed in a solvent system of chlorofonnzmethanol (1:1) and visualized
with 4(p-nitrobenzyI)-pyridine reagents. The slowest migrating compound (Rf
value of 0.33) gave the most intense spot of the three. Alter analyzing the
derivatives with FAB-MS methods, the authors concluded that the three
compounds (Rf values of 0.33, 0.60, and 0.68) were dihemisucciniC-DON, 3-
hemisuccinic-DON, and other isomer of hemisuccinic, respectively. These

derivative compounds were then conjugated to BSA and used as immunogen.

Antibody production

Despite use of Huntefs and Freund’s adjuvants and intrasplenic,

intraperitoneal, subcutaneous and intramuscular immunization techniques, all Of 20

141

 

"-3

 

142
female BALBIC mice and 6 female New Zealand rabbits that were immunized with

BSA-HS—DON conjugate were not able to produce antisera that could be used in a
competitive ELISA for DON. Difficulty producing DON antibodies was also reported
by several authors. Zhang et al. (1986) were also unsuccessful in producing DON
antibodies after immunizing rabbits with BSA-3-O-hemisuccinyI-DON conjugate as
well as with BSA-8-O-0arboxymethyl-0xime-DON conjugate (Figure 3.4). Initially,
Usleber et al. (1994) were also unable to produce DON antibodies alter they
immunized rabbits, mice and guinea pigs with human serum albumin-8-O—
carboxymethyl-oxime-DON conjugate and keyhole limpet hemocyanin-hydrazone-
DON conjugate. However, alter injecting three female Chinchilla bastard rabbits with
human serum albumin-HS-DON conjugate, Usleber et al. (1994) found one rabbit
which produced specific DON antibodies. Thus, it is possibly antibodies to DON may
be produced in rabbits other then the New Zealand White strains.

Previously, Casale et al. (1988) successfully produced DON monoclonal
antibodies after they immunized mice with BSA-3-HS-DON which was derivatized by
protecting C7 and 015 during esterification with succinic anhydride (Figure 1.5). It
seemed that the conjugation site to the carrier protein, rather than the immunization
techniques (intrasplenic, intraperitoneal, subcutaneous, or intramuscular
immunizations) was very critical for the production of DON specific antibodies. Thus,

the results of this study did not support this my hypothesis.

CONCLUSION

DON derivatives having epoxy and keto functional groups were readily
prepared by reacting DON with hemisuccinate. After a reaction of eight hours, DON
was completely converted to its derivatives. These toxin derivatives were
conjugated to BSA, and then emulsified with Hunter’s or Freund’s adjuvants prior to
animal immunization. Both adjuvants could induce the production of high titer
antisera. However, these antisera could not be used in competitive ELISAS for DON
and did not cross react with either 3-acetyI-DON, 15-acetyl-DON, nivalenol, 4,15-

diacetylnivalenol or fusarenone-X.

143

144

 

 

 

 

 

 

 

 

 

1.4 ——
; O lP-100 -.
1.2 — 3
i 0 lP-50 T
)- -<>—
1.0 —1 A sc-100
g“ I
,,, 0.8 4
c L
3. _
D: L
o 1-
0.6 a
0.4 —
0.2 —
C
0.0 1 1111111 1 I 111111 I 111L111 1 1 1L1111 1 1 I
10‘ 10‘ 10" 10:2 10'1

ANTISERUM DILUTION

Figure 3.1. Indirect ELISA titration of mouse antibodies obtained ten days after
the third injection with BSA-HS-DON conjugate. The first injection
was given at a dose of 50 and 100 pglmouse. The second and third
injections were given at a dose of 25 and 50 pglmouse. Each data
point represents the mean :I: standard error of the mean (n = 6,
duplicate measurements from three mice). IP indicates peritoneal
injection. SC indicate subcutaneous injection. 50 and 100 indicate
dose (pg) of the first injection.

 

W‘s

145

 

1.2

TTIV

1.0 —

fl I I I

08
O

IiTI I

06
I

0.0. (405 NM)

0.4 m

I I I I

0.2 -

I I I I

 

 

. SI-IP

‘ SI-SC 5
O TI-IP T

A TI-SC J

 

O /
I .11.

11111 1 1111111 1 1 1111111

 

 

 

 

 

Figure 3.2.

1 1
10‘1 10:2 10'1
ANTISERUM DILUTION

Indirect ELISA titration of mouse antibodies Obtained ten days after
the second (SI) and third immunizations (T I) with BSA-HS-DON
conjugate. The first immunization was given via intrasplenic
deposition at a dose of 20 pglmouse. The second and third
immunization were given via intraperitoneal (IP) and subcutaneous
(SC) injections at a dose of 10 and 50 p9, respectively. Each data
point represents the mean 1: standard error of the mean (n = 8,
duplicate measurements from four mice).

 

 

146

 

1.2

I I I I

1.0 —

I I I I

on
I

I I I I

06—
O

0.0. (405 NM)

I I I I

0.4 -1

I I I I

0.2 —

I I I T

 

0 HUNTER‘S ADJUVANT

O FREUND'S ADJUVANT

 

 

 

 

 

 

 

 

 

0.0

Figure 3.3.

104 104
ANTISERUM DILUTiON

Direct ELISA titration of rabbit antisera. Hunter's adjuvant indicates
that rabbits were given a two sites intramuscular injection with an
emulsion of Hunter Titer Max and BSA-HS-DON conjugate at a dose
of 100 pg per rabbit for both the first and second injections. Freund’s
adjuvant indicates that rabbits were given a ten site subcutaneous
injection with an emulsion of Freund’s adjuvant and BSA-HS-DON
conjugate at a dose of 500 pg and 250 pg per animal for the first and
second immunization, respectively. Antisera were obtained four
weeks after the second injection with BSA-HS-DON conjugate. Each
data point represents the mean 1 standard error of the mean (n = 6,
duplicate measurements from three rabbits).

maxi——

 

147

 

 

 

 

 

Compound R1 R2 R3 R4 R5
DON OH H OH OH =0
A O-HS-BSA H OH OH =0

8 OH H OH OH -O-CMO-BSA

Figure 3.4. Deoxynivalenol (DON) and its conjugates. BSA-3-O-hemisuccinyl-
DON (A) and BSA-8-O-carboxymethyl-oxime-DON (8). BSA, HS,
and 0M0 indiwte bovine serum albumin, hemisuccinyl, and

carboxymethyl-oxime, respectively.

148

 

PART IV.

PRODUCTION OF ANTIBODIES
AGAINST TRICHOTHECENE-YEAST RIBOSOMAL BINDING SITE

 

ABSTRACT

PRODUCTION OF ANTIBODIES
AGAINST TRICHOTHECENE-YEAST RIBOSOMAL BINDING SITE

31/

Sutikno

Both pure 608 and 808 ribosomal subunits were isolated from trichodennin-
sensitive yeast and used for the production of antibodies against trichothecene
ribosomal binding site. Twelve female BALBIC mice were immunized with either the
608 or 808 ribosomal subunits. Another ten female BALBIC mice were injected with
the mixture of the 808 subunits with different adjuvants. All mice apparently
produced high titer antisera. These antisera were used to develop a competitive
indimct enzyme-linked immonosorbent assay (CI-ELISA) for detection of
trichothecenes. However, this Cl-ELISA could not detect trichothecene because the

antisera were not specific to the trichothecene ribosomal binding site.

149

 

 

INTRODUCTION

Trichothecene mycotoxins

Trichothecenes are a group Of structurally similar sesquiterpenoid metabolites
produced mainly by toxigenic strains of Fusarium, and by some toxigenic strains of
Trichothecium, Trichoderma, Acremonium, Cylindrocarpon, Myrothecium and
Stachybotrys (Bamburg, 1983; Ueno, 1983; Committee on Protection Against
Mycotoxin, 1983; Betina, 1989). These mycotoxins, especially deoxynivalenol
(DON) and nivalenol (NIV) are found as natural contaminants in agricultural
commodities throughout the world (T anaka et al., 1988a). The presence of DON and
NIV trichothecenes in cereal and cereal products in several countries has been
surveyed and the results were summarized in Table 1.3. Trichothecenes can cause
a wide range of toxicoses to both animals and human (I' able 1.4) and cause large
economic losses to farmer and livestock producers (Table 1.1).

’ The presence of trichothecenes in agricultural products in the field and during
storage is mainly dictated by environmental factors and less importantly by genetic
factors (Figure 1.1). Controlling the Climatic and biological factors that contribute to
the presence Of the mycotoxins in agricultural commodities is almost impossible
(Pestka, 1988). Thus, detection and diversion are the most important means for

preventing the mycotoxin entry into food chains.

150

151

Immunoassay methods to detect the presence of mycotoxins in agricultural
commodities have recently gained wide acceptance because of sensitivity,
specificity, simplicity, and rapidity (Chu, 1991; Pestka et al., 1994a). lmmunoassays
for individual trichothecenes, such as T-2 toxin, DON, diacetoxyscirpenol, NIV, and
roridin A, have been developed to date (Table 1.5). Immunoassay kits for T-2 toxin,
and DON have also been produced and marketed in the United States (Table 1.6).
However, immunoassays that are capable Of detecting trichothecenes as a group in

agricultural commodities have not yet developed.

Ribosomal binding as a toxicity mechanism

Trichothecenes are potent inhibitors of eukaryotic protein synthesis (Bamburg
and Strong, 1971) because they bind to a common site on the 608 ribosomal subunit
(Carrasco et al., 1973; Barbacid, and Vazquez, 1974; Cundliffe et al., 1974;
Schindler et al., 1974; Cannon et al., 1976; McLaughlin et al., 1977). Occupancy of
this binding site by some trichothecenes, such as T-2 toxin and NIV results in
inhibition of protein synthesis at an initiation stage (Smith et al., 1975), whereas other
trichothecenes including trichodennin lead to block elongation and termination _
stages by interfering with the peptidyl transferase on the ribosomes (Cundliffe et al.,
1974)

In order to elucidate the binding site, Schindler et al. (1974) induced
mutations in a trichodennin-sensitive yeast strain (Saccharomyces cerevisiae
A224A) and then isolated a trichodennin-resistant strain (S. cerevisiae CLP-1). By

comparing the ribosomal components from both strains, these authors reported that

 

 

152

a mutant, which was resistant to the action of trichodennin, has an altered
component on its 608 ribosomes. The gene determining the altered 608 ribosomal
component is a single, recessive nuclear gene located on the right arm of
Chromosome XV and named tcm1 (Grant et al., 1976). By Cloning yeast genes for
trichodennin resistance and ribosomal protein L3 (the largest ribosomal protein),
Fried and Warner (1981) isolated tcm1 gene and found that the gene coded for
ribosomal protein L3. The nucleotide sequence of this tcm1 gene (ribosomal protein
L3) has also been determined (Schultz and Friesen, 1983).

The binding characteristics between trichothecenes and eukaryotic ribosomes
have been studied by several investigators. Jimenez and Vazques (1975) used
[acetyl-“0] trichodennin to study quantitative binding of trichodennin to ribosomes
from trichodennin sensitive(Y133) and trichodennin resistant (T R1) yeasts. They
found that Ym ribosomes bound to trichodennin with a dissociation constant (Kd) of
0.99 pM while those from the resistant one (T R1) bound to trichodennin with a Kd of
15.4 pM. Using similar techniques, Barbacid and Vazquez (1974) found that
trichodennin bound to the peptidyl transferase center of trichodennin-sensitive yeast
ribosomes with a Kd of 1.8 pM. and to 60$ subunit of the yeast ribosome with Kd Of
1.4 pM. Cannon et al. (1976) studied the binding affinity of the same toxin to
different states Of the sensitive yeast ribosomes (polyribosome and “run Off”
ribosome). They found that the binding affinity between trichodennin and the “run Off

ribosome (Kd = 0.72 pM) is approximately threefold higher than that between

trichodennin and the polyribosome (Kd = 2.1 pM).

 

153

Factors. affecting binding of trichothecenes and eukaryotic ribosomes have
also been studied. Changes in ionic concentrations (from 0 to 300 mM of
ammonium ion; from 0 to 25 mM of Mng), pH (from 6 to 8), and ethanol
concentration (from 0 to 40% vol.lvol) do not significantly affect the binding between
trichodennin and the peptidyl transferase center of yeast ribosomes although the
activity of the center is much affected by ionic concentrations (Barbacid and
Vazquez, 1974). Time and temperature of the reaction greatly affect the binding
between T-2 toxin trichothecene and the yeast ribosomes (Middlebrook and
Leathemnan, 1989). When incubated at 37°C, the association or 3H-T-2 with
ribosome is biphasic. In the initial phase, binding is rapidly increased and by 30
minutes a plateau equilibrium state is achieved. After that a second phase occurs in
which the binding gradually decreases to the base line value at a half-time of 2.5
hours. However, 3H-T-2-n'bosome binding at 4°C is much slower, taking 24-28 hours
to reach completion, and very stable, requiring 3 weeks to decay 50%. When T-2 is
prebound to ribosomes at 4°C and then incubated at 37°C, a significant degradation
process begins after a time lag of approximately 4 hours (Middlebrook and
Leathennan, 1989).

A ribosome has only one binding site and can bind to only one molecule of
trichothecene (Barbacid and Vazquez, 1974; Middlebrook and Leatherrnan, 1989).
Trichothecenes as well as anisomycin compete with each other for the ribosomal
binding site (Barbacid and Vazquez, 1974; Cannon et al., 1976; Middlebrook and

Leatherrnan, 1989). Interestingly, competition among trichothecenes for the binding

 

«I...

 

154

site appears to be proportional to their toxicity (Committee on Protection Against

Mycotoxin, 1983; Middlebrook and Leathennan, 1989).

Rationale and objective

Trichothecenes bind specifically to yeast ribosomes. These toxins compete
with each other for the ribosomal binding site in proportion to their toxicity.
Production of antibodies specific to the binding site would enable researchers to
develop ELISAs which an assess total “trichothecene load” in agricultural
commodities and , thus would enhance food safety.

Maximum binding between trichothecenes and yeast ribosomes is achieved
by 30 minutes when incubated at 37°C. Thus, developing indirect ELISA using
yeast ribosomes for coating microtiter plates (Figure 4.1) might be feasible. In such
an assay, trichothecenes and trichothecene-ribosomal binding site antibodies would
be incubated together over a solid-phase ribosomal binding site. Trichothecenes
would compete with the antibodies for the ribosomal binding site. The total bound
antibodies could be then detected by a second antibody which has been labeled
with an enzyme. After the addition of an appropriate enzyme substrate, the bound
antibody could be calculated based on the intensity of color development which is
measured spectrophotometrically. The “trichothecene load” would be inversely
related to a complex of bound enzyme-labeled antibodies.

Based on the above literature study, I hypothesized that antibodies against
trichothecene-yeast ribosomal binding site could be generated by immunization mice

with either 608 or 808 yeast ribosomes. The specific Objectives of this study were to

 

 

 

155

produce antibodies against trichothecene-yeast ribosomal binding site and use

these antibodies to develop indirect ELISA for trichothecenes as a group.

MATERIALS AND METHODS

Chemicals and reagents

All organic solvents and inorganic chemicals were of reagent grade or
better. Trichodennin sensitive S. cerevisiae (ATCC 46913) was obtained from
American Type Culture Collection (Rockville. Maryland). This strain was maintained
at 30°C on YEPD plates and grown in liquid medium at this temperature. Liquid
YEPD media were prepared by autoclaving of a mixture solution of 10 9 of yeast
extract (Difco Laboratories, Detroit MI), 20 g of bactO-peptone (Difco 0118, Difco
Laboratories, Detroit MI), and 20 g of dextrose in 1 liter of distilled water. Ovalbumin
(OA) (Chicken egg albumin grade III; fraction VII), Tween 20, 2,2'-azinobis(3-
ethylbenzthiazolinesulfonic acid)(A8TS), hydrogen peroxide, T-2 toxin, and
dithiothreitol were purchased from Sigma Chemical CO. (St. Louis, MO). Goat
anti-mouse immunoglobulin G (lgG)-horseradish peroxidase conjugate was
obtained from Cappel Laboratories (West Chester, PA). Tritium labeled T-2 toxin
(3H-T-2) was kindly supplied by Dr. Fun Sun Chu (University of Wisconsin,
Madison, Wisconsin). Methofane (methoxyflurane, inhalation anesthetic for

veterinary) was purchased from Pitman-Moore Inc. (Mundelein, IL).

156

1 57

Yeast production

Yeast was grown by the procedure of Treadgill et al. (1986). Briefly, colonies
were picked from plates and inoculated into several 10 ml liquid YEPD media
(cultures). After 10-18 hours of growthxfour of these cultures were used to inoculate
four separate 150 ml cultures, which were incubated at 30°C with slow
(approximately 150 rpm) shaking overnight. These cultures were examined
microscopically for possible bacterial contamination and two were selected and used
to inoculate two separate two liter cultures. These cultures were incubated at 30°C
with slow shaking until the cell solution reached an A333 of 2.0. The grown culture
was then cooled slowly to 8°C to induce ribosome run-Off. Cells were harvested and

washed twice in distilled water with centrifugation at 10,000 g for 5 minutes at 2°C.

Ribosome preparation

Yeast ribosomes were prepared by the procedure of Battaner and Vazquez
(1971) with slight modifiwfion. Briefly, each yeast preparation (approximately 100 9
wet weight of yeast cells) was resuspended in 300 ml of buffer containing 100 mM-
TriS/HCI. pH 7.4, 12.5 mM-magnesium acetate, 80 mM KCI, and 5 mM dithiothreitol.
Cells were then disrupted by four passages through a French Pressure Cell
(Aminoo) at 700 lbirrn2 (48.3 MPa), and the lysates were centrifuged at 20,0009 for
15 minutes at 2°C. The supernatant fractions were then centrifuged again at
20,0009 for 30 minutes at 4°C to Clear any remaining debris and kept at 4°C before

ultracentrifugation.

 

158

Clean supernatant fractions were sedimented by centrifugation in a Sorvall
RC-60 ultracentrifuge (DU Pont Company, \Nlllington, Delaware) using T865.1 rotor
at 45,000 rpm (150,0009) for 120 minutes. For washing, the pellet (crude
ribosomes) was suspended in 24 volumes of washing buffer (10 mM Tris-HCl buffer,
pH 7.4, 500 mM NH4CI, 100 mM Mg acetate, 5 mM dithiothreitol). An aliquot (5 ml)
Of ribosomes was layered on 6 ml Of a discontinuous sucrose gradient in washing
buffer (3 ml 20%, w/v sucrose in the bottom and 3 ml 5%, w/v in the top) and then
sedimented by centrifugation at 150.0009 for 8 hours. The ribosome pellet was
resuspended in about 25 volumes Of the standard buffer (10 mM Tris-HCl buffer, pH
7.4, 50 mM ‘NH4CI, 5 mM Mg acetate, 5 mM dithiothreitol). After centrifugation at
10,0009 for 10 minutes, the pellet was discarded, and the supernatant containing the
808 ribosomes was finally adjusted with the standard buffer to give a concentration
of approximately 30 mg ribosomes/ml, aliquoted, and stored at -80°C.

For preparation of 608 ribosome subunits, 1 ml Of the 808 ribosomes was
dialyzed for 2 hours against two liter of dialyzing buffer (10 mM Tris-HCI buffer, pH
7.4, 50 mM NH4CI, 0.2 mM magnesium acetate, 5 mM dithiothreitol) at 4°C. Linear
sucrose gradients were prepared by dissolving sucrose in the dialyzing buffer and .
slowly layered into 11-ml tubes of the T8651 rotor (Du Pont Company, Willington,
Delaware). The linear gradient consisted of 1 ml of each (from bottom to top) 25%,
20.5%, 17.0%, 13.0%, 9.5% and 7% sucrose (% sucrose was measured with
ABBEL-3L refiactometer, Milton Roy 00., Rochester, NY). Three ml of 4% sucrose
in dialyzing buffer, 1 ml of ribosome solution (15 mglml), and then 1 ml of the

. dialyzing buffer were gently layered onto this linear gradient.

159
After 4 hours of centrifugation at 122.0009 (40,000 rpm of the T8651 rotor),
the supernatant was fractionated and their absorbency was measured using
Spectronic 601 spectrOphotometer (Milton Roy 00., Rochester, NY). The pellet
(containing enriched 60$ subunits) was resuspended in the dialyzing buffer (5 ml)
and centrifuged at 101,8009 for 3.5 hours. The resultant supernatant was discarded,
and the 608 subunit pellet was rinsed and resuspended in standard buffer, aliquoted

and stored at -80°C.

T-2 toxin-ribosome binding assay

Ethanol precipitation techniques used to determine T-2 toxin—ribosome
binding were performed based on the procedure of Femandez-Munoz et al. (1971)
with slight modifimfion. Briefly, 100 pl of ribosome solution (0.4 pg ribosome/ml of 10
mM Tris-HCl buffer, pH 7.4, 50 mM NH4CI, 5 mM Mg acetate, 5 mM dithiothreitol)
was mixed with 10 pl Of standard buffer (10 mM Tris—HCl buffer, pH 7.4, 50 mM
NH4CI, 5 mM Mg acetate, 5 mM dithiothreitol) and 10 pl of 3H-T-2 solution (0.2
pCilml). After incubation at room temperature for 30 minutes, this mixture was mixed
with 60 pl precooled absolute alcohol and then incubated at 4°C for 30 to 90
minutes. Ribosome-bound 3H-T-2 was separated from the solution by centrifugation
at 30009 for 20 minutes at 4°C. One hundred (100) pl of the supernatant was
carefully removed and mixed with 4 ml Of scintillation cocktail (Research Products
lntemational Corp.) into scintillation vials. Radioactivity was determined by liquid
scintillation counting using a Packard TRICARB 4430 Liquid Scintillation System

(Packard Instrument InC., Downers Grove, IL).

 

 

160

The resUltant value provided an estimate for the concentration of the labeled
toxin in free solution at equilibrium. For control, the total radioactivity was
determined in dupliwte samples in which ribosomes were omitted. The difference
between the total radioactivity and the radioactivity in free solution gave an estimate
for the concentration of ribosome-bound compound.

Competitive binding assay was used to determine whether nonradiolabeled
T-2 toxin or mouse sera competed with tritium labeled T-2 toxin for trichothecene-
ribosome binding site. Briefly, 100 pl of ribosome solution (0.4 pg ribosome/ml of
standard buffer) was mixed with 10 pl of serially diluted (0-1000 nglml) T-2 toxin
standard solution or 10 pl of appropriately diluted mouse sera. After incubation at
room temperature for 30 minutes, 10 p1 Of 3H-T-2 solution (0.2 110le) was added
and the mixture was incubated for another 30 minutes at room temperature. The

binding assay was then completed as described above.

Mouse immunization

Twelve female BALBIC mice (6 to 8 weeks of age, Charles River
Laboratories, Wilmington, MA) were immunized by intraperitoneal (i.p.) and
subcutaneous (s.C.) routes. The mice were immunized four times with an
emulsion of 608 or 808 subunit of yeast ribosomes with Freund’s adjuvant at a
ratio of 1:1 (vol.lvol.) at two-week intervals. The first immunization was
performed at week zero at a dose of 50 p9 ribosome/mouse and the ribosome
was emulsified with Freund’s complete adjuvant. The second, third and fourth

injections were performed at week 2, 4 and 6 at a dose of 25 p9

 

 

161

ribosomes/mouse and Freund’s incomplete adjuvant was used to emulsify the
ribosomes. Ten days after the second, third and fourth immunizations,
methoxyflurane-anesthetized mice were bled from the tail vein and the blood was
collected with a heparinized tube. The blood was incubated at 4°C overnight and
then centrifuged at 10009 for 15 minutes to Obtain mouse plasma. Antibody titer

and specificity were then determined by indirect ELISAS as described below.

Indirect ELISA.

Indirect ELISA was performed by a modification of the procedure of
Azcona-Olivera et al. (1992a) and used to determine serum titers. Briefly, wells
of polystyrene microtiter plates (lmmunolon 4, Dynatech Laboratories, Alexandria,
VA) were coated overnight (at 4°C) with 100 pl of ribosomes (5 pglml) in standard
buffer (10 mM Tris-HCl buffer, pH 7.4, 50 mM NH40l, 5 mM Mg acetate, 5 mM
dithiothreitol). Plates were washed four times by filling each well with 300 pl of the
standard buffer and aspirating the contents. Nonspecific binding was blocked by
filling the wells with 300 pl of 1% (wt/vol) ovalbumin in the standard buffer. After
incubating for 30 minutes at 37°C, the plates were washed four times with the
standard buffer. Fifty pl of serially diluted mouse serum was added to each well
and incubated at 37°C for 30 minutes. Wells Of serially diluted preimmune serum
were used as control. Unbound antibodies were removed by washing four times
with the same buffer and 100 pl of goat anti-mouse IgG peroxidase conjugate (2
pglml of the ovalbumin solution) was added to each well. The plates were

incubated for 30 minutes at 37°C, washed eight times with the standard buffer

 

 

162

and then rinsed twice with distilled water. Bound peroxidase was determined with
ABTS substrate [1 ml of 35 mg 2,2’-azinobis(3-ethylbenzthiazolinesulfonic
acid)/15 ml distilled water mixed with 11 ml of Citrate buffer pH 4 and 8 pl
hydrogen peroxide] as described previously by Pestka et al., (1982). Absorbance
at 405 nm was read with a Vmax Kinetic Microplate Reader (Molecular Devices
Corporation, Menlo Park, CA). Titer of each serum was arbitrarily designed as
the maximum dilution that yielded twice or greater absorbance as the same
dilution nonimmune control serum.
A competitive indirect ELISA (Cl-ELISA) was used to test the potential for
T-2 to block the binding of antibody to yeast ribosomes. verify specificity of
antibodies in sera toward T-2 toxin. Briefly, microtiter plates were coated and
blocked as described in the indirect ELISA procedure, and then 50 pl of serially
diluted (0-10,000 nglml) T-2 standard solution was simultaneously incubated with
50 pl of appropriate dilution of antibodies over the ribosome solid phase for 30

minutes at 37°C. The assay was then completed as described above.

 

 

RESULTS AND DISCUSSION

Ribosome preparation

Four liter yeast cultures were prepared by inoculating trichothecene-
sensitive yeast into 4 liter liquid medium and then incubating it at 30°C with slow
shaking for one week. These cultures were harvested by centrifugation at
10,0009 for 5 minutes and resulted in approximately 80 g wet weight yeast cells.
From these, approximately 50 m9 of 808 ribosomal subunit and 30 mg of 608
ribosomal subunit were prepared. RiboSomal purity was checked by calculating
the values of ribosomal absorbance at 260 nm (A233)divided by that at 280 nm
(A233). The A233! A233 values of the 808 and 608 ribosomal subunits were 2.08
and 1.95, respectively, indicating that the ribosomal subunits were pure since
proteins including ribosomes are considered as pure or uncontaminated if the
value ofA2331 A233 is greater than 1.80 (Spedding, 1990).

Tritium labeled T-2 toxin was used in ribosome binding assays. 3H-T-2 was
incubated with yeast ribosomes diluted in the standard buffer. As a control the same
amount of the radiolabeled toxin and the standard buffer were mixed without the
ribosomes. After separation of ribosome-bound 3H-T-2 by centrifugation,
radioactivity Of the supernatant was measured. It was found that supernatant from
ribosome supernatant had a lower radioactivity values (disintegration per minute,

DPM) than that fiom the control solution indicating that some of the 3H-T-2 bound to

163

164

the ribosomes. This suggested that the trichothecene—ribosomal binding site was still
active. When these ribosomes were reacted with the radiolabeled toxin in the
presence of free T-2 toxin, the association of the 3H-T-2 to the ribosomes was
inhibited by the free toxin (Figure 4.2). These ribosomes, which still had the

capability to bind to T-2 toxin, were used to immunize female BALBIC mice.

Mice immunization

Initially, twelve mice were immunized and boosted intraperitoneally and
subcutaneously with an emulsion of 808 or 608 yeast ribosomal subunits and
Freund’s adjuvant. Ten days alter the booster injection, the mice were bled. The
blood was incubated at 4°C overnight and then centrifuged at 10009 for 15 minutes
to obtained mouse plasma. The mouse antisera were analyzed by indirect ELISA to
determine their titers and specificities. These antisera had high titers (Figure 4.3)
indicating that they could recognized solid phase bound ribosomes. However, these
antibodies could not inhibit the association of trichothecenes to yeast ribosomes
when used in Cl-ELISA. In addition, when these antibodies were incubated together
with ribosomes and tritium labeled T-2 toxin during binding assay, they could not
inhibit the association of the radiolabeled toxin to the ribosomes. Thus, these
antibodies were not applicable for assays of “trichothecene load” in food samples.

In subsequent experiments, ten mice were immunized subcutaneously (SC)
using 803 ribosomal subunit because in the previous experiments, SC immunization
yielded higher titer antibodies (Figure 4.3). Percent inhibition of T-2 toxin to the

association of tritium labeled T-2 toxin to 808 ribosomal subunit was similar to that to

 

 

 

 

165

the 608 one (Figure 4.2). Moreover, preparation of the 808 subunit was much
simpler than that of the 608 one. In this immunization, the 808 ribosomal subunit
was mixed either with T-2 toxin (T -2—80$), Freund’s adjuvant (FA-808), Cholera toxin
(CT-80$), or with a mixture of FA and CT (FACT-808) prior to animal immunization.
All mice apparently produced antisera with high titers (Figure 4.4). However, these
antisera could not inhibit the reaction Of trichothecenes and yeast ribosomes when
they were used either in competitive indirect ELISA or in binding assays.

Animal immunizations with 808 yeast ribosomal subunits either alone or
mixing with adjuvants could not induce the production Of antisera that could be
utilized in competitive inhibition ELISA for trichothecenes. This may relate to two
possibilities. Firstly, trichothecene-ribosomal binding site might be modified during
immunogen preparation or during metabolism in animal body, and thus antibodies
that were specific to the binding site were not generated. Secondly, the antibodies
specific to the binding site might have been produced by the immunized animals but
in a small proportion as compared to non specific polyclonal antibodies. As a result,
trichothecene inhibition to the specific antibodies were undetectable when these
antisera were used in CI-ELISA for trichothecene detection. Thus, these results did '
not support the hypothesis of this current study.

Direct injection of specific proteins or specific oligopeptides for the binding site
into animals, rather than “whole” ribosomes, might be capable to stimulate the
desired specific antibodies against the binding site. The specific proteins for
trichothecene-ribosomal binding site could be isolated by comparing ribosomal

components from trichodennin-sensitive yeast to that from trichodennin-resistant

166

one. Schindler et al., (1974) found an alteration of 608 ribosomal subunit in the
trichodennin-resistant mutant. The alteration was associated with ribosomal protein
L3 which was coded by tcm1 genes (Grant et al., 1976; Fried and Warner, 1981).
Thus, injection ribosomal protein L3 from trichodennin-sensitive yeast into animals
may be as an alternative to produce antibodies against the ribosomal binding site.
Another alternative is immunization of specific oligopeptides for the binding site.
The nucleotide sequence of tcm1 gene that encodes ribosomal protein L3 in
trichodennin-resistant yeast strains has been determined (Schultz and F riesen,
1983). By comparing this nucleotide sequence and the nucleotide sequence of a
gene encoding protein L3 In trichodennin sensitive yeast strains, specific nucleotide
sequence for trichothecene-ribosomal binding site could be isolated. From this
sequence, polypeptides could be constructed in vitro and might be used for the
production of specific antibodies against trichothecene-yeast ribosomal binding site.

 

 

 

 

CONCLUSION

Both pure 608 and 808 ribosomal subunits have been isolated from
trichodennin-sensitive yeast and used for the production of antibodies which were
specific to trichothecene ribosomal binding site. These ribosomal subunits (either
alone or after mixing with different adjuvants) were used to immunize 22 female
BALBIC mice. All mice apparently produced high titer antisera. However, these
antisera were not able to inhibit the association of trichothecenes and yeast
ribosomes when they were used either in Cl-ELISAs or in binding assays for
trichothecenes. Thus, the production of antibodies that were specific to
trichothecene-ribosomal binding site could not be accomplished through animal

immunization with either 608 or 808 subunits of yeast ribosomes

167

 

 

168

 

 

 

Figure 4.1. Competitive indirect ELISA for “trichothecene load” using yeast
ribosomes for coating microtiter plates. Trichothecene ribosomal
binding site specific antibodies (AB) compete with trichothecenes (T)
for the trichothecene binding site (TBS). Second antibodies (SAB)
which have been labeled with enzyme (ENZ) are then added to
determined total bound antibody (AB). ”Trichothecene load” is
inversely related to the bound enzyme-labeled antibodies and can be
measured quantitatively using spectrophotometer after color
development by the addition Of enzyme substrate.

169

 

110

100 -

so

00%

70—

50—

40—

PERCENT INHIBITION

30—

20—

 

 

    

. 803 RIBOSOMAL SUBUNIT

  

  

A 603 RIBOSOMAL SUBUNIT

 

 

I I I I T I I I I I I

-100 0 100 200 300 400 500 600 700 000 900 1000 1100

Figure 4.2.

T-2 TOXIN (nglml)

Inhibition of T-2 toxin to the association of °H-T-2 toxin to yeast
ribosomes. Each data point represents the mean value of triplicate
measurements. One hundred pl of yeast ribosomes (0.4 mglml
standard buffer) were reacted with 10 pl of 3H-T-2 toxin (0.2 uCi/ml)
in the presence of 10 pl -of serially diluted (0 to 1000 ng) non
radiolabeled T-2 toxin and 60 pl of precooled alcohol. As a control
the same amount of radiolabeled toxin was mixed with the standard
buffer without ribosomes. After ribosome-bound 3H-T-2 toxin was
separated by centrifugation, radioactivity (DPM) of 100 pl of the
supernatant was measured by liquid scintillation counting. DPM values
Of control solution and 0 nglml supernatant were 16,026 and 8,577,
respectively. Percent inhibition = [(DPM value of certain n9 T-2 toxin
lml supernatant - DPM value of 0 nglml supematant)l(DPM value of
control solution - DPM value of 0 nglml supematant)] x 100%.

170

 

 

 

 

 

 

 

 

 

2.0
)—
1-3 -_ 0 sc-80s
Z O lP-80s
. —” ~ 1 .
1 ° - A sc-sos
I A IP-sos
1.4 —- _
E : --
.3 1.2 —
3. :
d. :
0 1.0 —
0.8 ---h “ 1
0.6 _‘- /‘\/
0.4 —- ‘
1 I L I 1 I 1 1 l 1 1 1 I 1 I
10‘1 103
ANTISERA DILUTION
Figure 4.3.

Typical indirect ELISA titration of mouse antisera. The mouse
antisera were obtained ten days after the third subcutaneous or
intraperitoneal injection with 50pg of 608 or 808 yeast ribosomal
subunit emulsified with Freund’s adjuvant. Each data point
represents the mean 1: standard error of the mean (n=6, duplicate
measurements Of three mice). IP, and SC indicate intraperitoneal and

subcutaneous injections, respectively. 808 and 608 indicates 808
and 608 yeast ribosomal subunits, respectively.

 

 

 

171

 

2.0

1.8 J D
i O

1.6 —

IIII

d

.

1
IVI TI
4\.

1.2 —“‘

I
80s ONLY
FA - 80s ' .
0 CT - 30$ /
1-

A FACT - 808

IITI

 

1.0 -

0.0. (405 nm)

rIII

08—
O

 

A T-2 - 008
0.6 —

 

IIjfiIITI

0.4 H

0.2 —

 

 

jIIIIIII

 

10‘ . 10*2
ANTISERA DILUTION

Figure 4.4. Typical indirect ELISA titration of mouse antisera. The mouse
antisera were obtained ten days after the third subcutaneous
injection with 50pg of 808 yeast ribosomal subunit mixed with
different adjuvants. Each data points represents the mean :I:
standard error of the mean (n=4, duplicate measurements of two
mice). 808 indicates 80$ yeast ribosomal subunit. FA, CT, and T-2
indicate F reund’s adjuvant, cholera toxin, and T—2 toxin, respectively.

APPENDICES

172

APPENDIX A:

MEDIA FOR HYBRIDOMA PRODUCING MONOCLONAL ANTIBODIES

1. Chemical and reagents

All organic solvents and inorganic Chemical were of reagent grade or
better. Penicillin/streptomycin solution (pen/strep) (100,000 units/ml), sodium
pyruvate, polyethylene glycol (MW 1450) (PEG), hypoxanthine, aminopterin,
thymidine, pristane, and dimethyl sulfoxide were purchased from Sigma Chemical
CO. (St. Louis, MO). Dulbecco’s Modified Eagle’s Medium (DMEM, in powder
form for 1 l medium/bottle), NCTC supplemental medium, and fetal bovine serum
(PBS) were Obtained from Gibco Laboratories (Grand Island, NY). Tissue culture
plasticware was purchased from Corning Laboratory Science 00. (Corning, NY).
The myeloma cell line P3INS1/1-Ag4-1 (NS 1) (ATCC TIB 18) was purchased
from the American Type Culture Collection (Rockville, MD). Macrophage

conditioned media (MCM) was prepared as described by Sugasawara et al.
(1985)

II. MEDIA
Dulbecco's modified eagle's medium (DMEM)

One liter Of distilled water was poured into a 2 liter Erlenmeyer flask.
Powder of DMEM was added to the flash and stirred with a magnetic rod until the
powder was dissolved. Sodium bicarbonate (3.7 gll) was weighed and added so
that the medium color Changed from orange to red. pH of the medium was
measured and adjusted to neutrality (pH 7.0) either with 1N NaOH or 1N HCI.
Usually the medium was slightly basic so that 4-5 ml of 1N HCI was added. Prior
to sterilization with filter sterilized (Nalgene disposable filter unit catalog number.
155-0020; volume: 150 mL; memb mat type: C.A./T.A; color code: yellow, pore
size: 0.2 mm), 10 ml Of NCTC suplement and 10 ml of sodium pyruvate were
added to the neutral medium so that the DMEM contained 1% NCTC and 10 mm
sodium pyruvate. The sterilized DMEM medium was stored in a sterilized and
labeled 500 ml bottle at 4°C and tested for its steril' by incubating two separte 1

ml of the medium into wells of a 24-well plate at 37 C in an incubator containing

5% 002 for 2 days. If the medium was contaminated by microorganims, the
medium has to be refiltered, and then rechecked for its sterility again.

173

Maintenace medium

Twenty ml of fetal bovine serum (FBS), and 1 ml of Pen/Strep (PIS) solution
were added to 80 ml of the sterilizied DMEM medium to make 20% PBS-DMEM.
The 20% FBS-medium was filter sterilized (Nalgene disposable filter unit catalog
number. 155-0020; volume: 150 mL; memb mat type: CAITA; color code: yellow,
pore size: 0.2 mm) and stored at 4°C.

One hundred x stock solution of hypoxanthine-thymidine (HT)

One hundred x stock solution of HT was prepared by suspending 166.1 mg
of hypoxanthine (H) and 38.75 mg of thymidine (T) in 50 mL of distilled water. The H
and T were dissolved by adding (0.1N or 1.0N) NaOH dropwise until H and T were
solubilized. The volume was adjusted to 100 ml with distilled water, filter sterilized,

aliquoted in 10 ml, and stored at -20°C.

Fifty x stock solution of HT

Fifty x stock solution of HT was prepared by adding an equal volume of 100
x Stock solution HT to an equal volume of the sterilized DMEM and stored at 4°C.

Aminopterin stock solution

Aminopterin stock solution was prepared by adding 17.6 mg of aminopterin
(A) to 100 ml of distilled water and stored at -20°C in aliquots of 10 mlltube.

Fifty x Stock Solution of HAT

Fifty x Stock Solution of HAT was prepared by adding 50 ml of 100 x stock
solution of HT and 5 ml of aminopterin stock solution to 45 ml of the sterilized
DMEM, filter sterilized, and stored at 4°C in aliquots of 12.5 mlltube.

HAT medium

HAT medium was prepared by adding 20 ml of PBS, 1.25 ml of PIS, and 2.4 ml of
50 x HAT stock to 100 ml of the sterilizied DMEM, filter sterilized, and stored at 4°C.

 

 

 

174
HT Medium

HT Medium was prepared by adding 20 ml of FBS, 1.25 ml of PIS, and 2.4

ml of 50 x stock solution of HT to 100 ml of the sterilized DMEM, filter sterilized, and
stored at 4°C.

Macrophage Conditioned Medium (MCM)

Five female BALBIC mouse (6 to 8 weeks of age, Charles River
Laboratories, Wilmington, MA) were sacrificed, dipped in 70% ethanol in water to
sterilize their whole bodies, and peeled back their skin to expose their peritoneal
lining. Ten ml of 20% PBS-DMEM was injected to mouse’s peritoneal cavity using
an 18 gauge needle. The peritoneal cavity was tapped several times to dissolve
macrophage cells into DMEM which was then drew off using the same needle. The
DMEM containing macrophage cells was centrifuged at 450 x 9 for 7 minuted. The
cells (sediment) were resuspend in 40 ml of 20% FBS—DMEM, and placed into two
large T-flasks (20 ml each). After incubation at 37°C in an incubator containing 5%
CO2 for 3 days, the medium was collected, and the cells were rated with 20 ml of

fresh 20% F BS-DMEM medium. The medium was harvested 2 more times at 3 day
intervals. The medium was stored at -20°C.

 

 

Ten or 20% macrophage-HT medium

Ten or 20% macrophage-HT medium was prepared by adding 10 or 20 ml
of MCM to 90 or 80 ml of HT medium, filter sterilized, and stored at 4°C.

Cloning medium

Cloning medium was prepared by adding 20 ml of MCM to 80 ml of 20%
FBS-DMEM, filter sterilized, and stored at 4°C.

Lysing buffer

Lysing buffer was prepared by adding 8.29 9 of NH4CI, 1.0 g of KHCO3 and

0.037 g of EDTA tO 1 liter of double distilled water, filter sterilized, and stored at room
temp.

175

APPENDIX B:

SAMPLE LISTS OF CORN AND CORN PRODUCTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5.1. Number and description of food samples bought in retail
supermarket in Mid Michigan, in September, 1991.
SAMPLE: DESCRIPTION
#
6 Self-rising white, corn meal
7 Yellow corn meal
8 Self-rising white, com meal mix
10 Yellow corn meal
21 Self-rising white corn meal
22 Self rising white corn meal mix
24 Corn tortilla mix
25 White yellow plain enriched corn meal
32 Yellow corn meal
33 White corn meal
38 Seven grain cereals
41 Corn cereals
43 Corn flakes
48 Corn meal
H9 Corn meal

 

 

 

 

 

 

 

176

Table 5.2. Number and description of Italian feed samples.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SAMPLE DESCRIPTION SAMPLE DESCRIPTION
NUMBER NUMBER
F1 Feed for goat F17 Feed for laying Chicken
F2 Feed for milk cow F18 Feed for swine
F3 Complementary feed for horse F19 Feed for swine (45% maize)
F4 Feed for calf F20 Starter feed for swine
F5 Feed for Chicken F21 Feed for swine
F7 Flour for pig F22 Prestarter feed for swine
F8 Feed for swine on phase Of growth F23 Feed for pregnant cow
F9 Feed for milk cow F24 Feed for Chicken
F10 Feed for swine F25 Silage maize
F11 Feed for milk cow F26 Feed for swine
F12 Flour of maize F27 Feed for laying chicken
F13 Feed for swine F28 Feed for swine
F14 Feed for milk cow F29 Feed for buffalo
F15 Feed for calf (50% maize, 50% F30 Feed for calf
barley)
F16 Feed for rabbit F31 Feed for calf

 

 

 

177

Table 5. 3. Number and description of fresh corn sample harvested from five
counties in Michigan in Summer, 1994a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SAMPLE NAME AGE (DAYS) LOCATION
NUMBER
P1 CaLgill 3777 ' 98 Montcalm
P2 Cagill 4327 104 Montcalm
P3 Dekalb 0K 527 102 Montcalm
P4 Pioneer 3293 1 13 Monroe
P5 Pioneer 3394 110 Montcalm
P6 Amcom 5930 110 Cass
P7 Callahan 07337 97 Cass
P8 Callahan C7446X 103 Cass
P9 Cargill 3777 98 Cass
P10 Carfll 7777 1 15 Cass
P1 1 Counflmard 432 100 Cass
P12 Dekalb 0K 471 97 Cass
P13 Dekalb 0K 560 106 Cass
P14 Mycogen 6060 107 Cass
P15 Peyco 614 98 Cass
P16 Pioneer 3293 1 13 Cass
P1 7 Pioneer 3525 1 06 Cass
P18 Renk 646PT 105 Cass
P19 Rusb XR-1727 106 Cass
P20 Callahan 07252 107 Huron
P21 Cargill 3777 98 Huron
P22 Cargill 4327 105 Huron
P23 Cargill 5547 106 Huron
P24 Country Mark 432 100 Huron
P25 Dairy cand Stealth 1205 105 Huron
P26 Decald 0K 471 97 Huron
P27 Dekalb 0K 527 102 Huron
P28 Mycogen 3440 93 Huron
P29 NK 4242 100 Huron
P30 Northrup King N K4242 100 Huron
P31 Peyco 614 98 Huron
P32 Pioneer 3394 1 10 Huron
P33 Pioneer 3525 106 Huron
P34 Renk RK 657 106 Huron
P35 Rupp XR 1727 106 Huron
P36 Amcorn 5930 1 10 Monroe
P37 Callahan 07337 97 Monroe
P38 Callahan C7446X 103 Monroe
P39 Cargill 7777 1 15 Monroe

 

 

1 78
Table 5.3. (Cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SAMPLE NAME AGE (DAYS) LOCATION
NUMBER

P40 Country Mark 432 100 Monroe
P41 Dekalb 0K 569 106 Monroe
P42 Dekalb OK 471 97 Monroe
P43 Mycogen 6060 107 Monroe
P44 Northrup King K4242 - Monroe
P45 Peyco 614 98 Monroe
P46 Pioneer 3525 106 Monroe
P47 Rank 646 PT 105 Monroe
P48 Rupp XR 1727 106 Monroe
P49 Cargill 3777 91 - Montcalm
P50 Cargill 5877 108 Montcalm
P51 Country Mark 432 100 Montcalm
P52 . Decald 0K 471 97 Montcalm
P53 Junflg2672 108 Montcalm
P54 Mycogen 3440 93 Montcalm
P55 NK 4242 100 Montcalm
P56 Peyco 614 98 Montcalm
P57 Pioneer 3394 1 10 Montcalm
P58 Pioneer 3525 106 Montcalm
P59 Rank 657 106 Montcalm
P60 Renk RK 657 106 Montcalm
P61 Rupp 1727 106 Moutcalm
P62 Callaahan 7446 x 103 Saginaw
P63 Callahan 07337 97 Saginaw
P64 Cargill 3777 98 Saginaw
P65 Cargfl 4277 102 Saginaw
P66 Cargill 6677 1 10 Saginaw
P67 Clba 4394 107 Saginaw
P68 Country Mark 432 100 Saginaw
P69 Dekalb 0K 471 97 Saginaw
P70 Dekalb 0K 569 106 Saginaw
P71 Mycogen 6970 107 Saginaw
P72 NK 4242 100 Saginaw
P73 NKX 423 105 Saginaw
P74 Peyco 614 98 Saginaw
P75 Pioneer 3394 1 10 Saginaw
P76 Rank 646 PT 105 Saginaw
P77 Rupp XR 1677 106 Saginaw

 

‘ kindly supplied by Dr. Patrick L. Hart (105 Pesticide Research Center, Michigan State
University, Ml, 48824.

 

APPENDIX C

IMMUNOLOGICAL ASSAYS FOR MY COTOXIN DETECTION

 

179

 

Immunological Assays for Mycotoxin Detection

Enzyme—linked immunosorbent assays have been success-fully applied
to the screening of mycotoxins in a diverse array of foods

James J. Pestka. Mohamed N. Abouzied. and Sutikno

 

D MYCOTOXINS ARE TOXIC SEC-
ondary metabolites produced by molds
that often contaminate agricultural stao
plea such as corn, wheat, and peanuts
rior .to harvest and during storage.
ese compounds have a wide array of
chemical structures and are produced by
common field and storage fungi, includ-

many s 'es of Aspergt'llus, Perri.

Mycotoxinscanelicitavariety oftosic
symptoms in humans and animals, -
ing from gastroenteritis to cancer (Tab e
1). For example, the aflatoxins were
identified in the early 19603 as etiologic
agents of hepatotoxicity and hepatic
cancer in hark poults and rainbow
trout. respects y (Pestka and Casale.
1990). Strrct regulations were subse-
quently established for aflatoxins in food
inmanycountriesbecsuseot’the poten-
tial for similar effects in humans.

Besides direct concerns over human
health. aflatoxins and other mycotoxins
have major economic impact on live-
stock productivity as a result of lower
quantrtyand quality of animal produgsd,
smaller litters, infertility, reduced 1'

' ' ' resistance to. dis-

Whetheram
foodielargelyd‘ixotaid“l
talandbiologicalfactorsfi’ig. 1).par-
tiarlarlytheregionalweatherduringa
means ehminatmgmycotonnstrmn
hmnansndanimalt’oodntodetectend

ntaminatedrswmaterialsfipm

 

 

 

 

 

 

 

 

Tabla I—Mejor Mycotoxins sndtheeroxlc Effectshemerirmrdanimdmodsb
Mycotoxin Tealeeflect M
AflsroeinsB..02.Gr.Ga Livsrtosicitysndcsnosr WWW.

m
AflatoaLiM. Liversoaicirysndcsnosr Milt
Cydooissonicsa’d Musdsnesrosimorsl Cerium
lesions
Fm l ‘ ., ' Com
pdmonsryedsms
Odrstoain WM.” monuments
cats
PM Medicaid" mumps}:
net-staid"
Tridmmirdufing Fesdreltrsstdermea. Madman-11w
Winni- orslerdGIIesions.
MuiMT-Z W
rosin
Zearalenone Mamas."- Column!"
Mutations
utnwheagcottonseed.andother mimosorbentassaymfl‘heseand
1 ssvrellasclinicalsamplestrom otheressaysheveheenmsdeavailable
htunansandanimalsAseriesot’esten- comm ' {fortherspidusayofmy-
sive cleanup steps involving liquid-liq- ootoxins in cod.
uidpartition.columncleanup,andevap- Key considerations in the
orationarethereforerequiredtoover- meatand ' ' 01' ' im-
come these interferences. '11: munoassaystot’oodmanalydsoin-
proceduresarstime-consmningand cludemethodot’an generation.
costlyandofteninvolveuseofharmful immunoassayt’ormats. to
solvents. food andysigandevalustion criteria {or
Researchinitietedinthelatelm oommercialtesthits.

roposed the application of immuno-
ghemical rocedures commonly
medindm toriestotheanal-
ysis of aflatoxin B; (AFB and other
mycotoxins (Chu and 1977;
LangoneandVanVunakisJWG .Theae
assaystypicallyinvolvethecompetition

between a free woman in a sample
extractant! a edmycotoxin {oran
antr’bodybindingsiteAl initially
basedonrsdioimmunoasay sub-
sequent research has established the
feasibility of using enzyme-linked im-

 

Author Pestka. a Professional Member d
[FT isProtessor.andauthorsAhoozisdand

' State
University.EastLansing.hfl48824.Sendre-
print requests to author Pestka.

 

Generation of Mycotoxin
Antibodies
Antibodieamrhnmmoglobulimarea
10:3 of glycoproteins that are pro-
d asa foreiflzolewles
in the ham and 1988).
This ' the interaction

responserequlres
among white blood cells (leukocytes)
knownasBcelIaToelhandmacro-
phagethatculminsteintheterminal
difl'erentiation of the B cells into anti-
body-eecretingplasmaoells.

muneresponseandisdepend
Chemicalstructnreanditsabilitytope
recognisedasforeignmaterialtlhemm-
imum moleatluweightofanrmmuno-
genis3,000-5,000. Because mycotoxins
are oflow molecular ’ ht (300409).
they must be earnings to a‘camer

180

 

 

Tabe—EconomiefiffectsofMyco—
tordncontamhsdcnhlood. W
MCASTHSOQI

 

Harder/miter

International trade

 

 

 

geroteinsuchasbovineserumalbuminto
Imm

Imogemc. .
Often. preparation of a suitable my-
cotoxin immunogen is the rate-limiting
step in the development of an immu-
noassay. Standard hapten conjugation
techniques used for mycotoxins have
been reviewed by Chu (1986). lithe my-
cotoxin doesn’thsve a reactive up for
conjugation. it must be derivatized. [for
example, generation of protein conju-
gate {or the mycotoxin fumonisin, (Asco-
na-Oliver'setal..19923.b)srm lym-
volves the use of glutaraldehyde °
conjugs o eoxynrval Casalee
al.. 1988) is much more dificult because
cchng’ stages (Fig. . nee-ally, e
same conjugation techniques used for

immrmcgen preparation can be applied

to link to to enzyme markers
forthe Mlongssthereaction
conditions do not denature the enzyme.
In some cases. undesirable side reac-
tions can occur during chemical conju-
'onandcanresultinantibodrestothe

- roducts (Gendlofl' et al., 1986). An-
‘es may also react with mycotoxin

plus bridge bridge group. plus
carrier protem, or the carrier protein di-
rectly. Thus, when ’ ° and
evaluating mycotoxin antibodies for im-
munoassay, conjugation reaction proto-

 

 

 

 

 

 

 

 

 

 

 

HARVESTING

 

 

 

 

 

 

 

an

 

Molsture

STORAGE
Temperature

Detection/Diversion

 

 

 

 

 

iii

HUMANS ;

 

 

 

 

59. I—FectorsAffectir-Iglllycosoxln Oeewreneshdrelcoddrshfiomfcaeandthsefe

(1990!

colsmustbecarefullyselectedandap—
propriate controls utilised.
. A key aspect in antibody development
18 the site of chemical conjugation For
example, many approaches have been
used to roduce antibodies with differ-
ent specrficities for the aflatoxin family
(F111. 3). Those portions of the aflatoxin
m enile which project distally from the
conjugation site are said to be immuno-
dorrrmont, became the resultant anti-
bodies will exhibit the highest degree of
recognition {or these moieties. Thus.
any metabohc precursor or analogues
which mimic this immunodominant re-
gronwillberecognisedbyanantibody
generated against the t toxin.
Cross-reactivity can assessed using
RIAorELISAcompetitioncur-veawhere
the levels required for 50 96 inhibition of
marker ligand ’ ' are as the
bastsofcomparjson ig.4).Rarelyare
these competrtron curves superimpos-
able; thus, an analogue typically cross
reacts to a greater or lesser extent than
the parent toxin. Although the presence
ofsuch analogues in a sample may ren-
der a quantitative ass to the level of
semiquantitative, a high level of cross-

reactivi can beveryuseful. ashasbeen
o for the screening of fumonisins
(Ascona-Olivera et al.. 1992a. b), zearale-
nones (Dixon et al., 1987; Warner and
Pestka. 1986) and the aflatoxins (Dixon
et al.. 1988).

The most straightforward approach
to generation of antibodies is the multi
pie-site immunization of rabbits with
too-1,000 pg of mycotoxin—protein con-
jugateUaa leantiserumcouldbeob—
tunedinHmdAaiticalfsctorinthis
immunizationproceaaistheuseofan
oil-based adjuvant MW
Mycobacterium such as the ’s
Complete type” to allow slow release of
the immunogen and nonspecifically
stimulate the immune nae. More
recently. we have utilized
very low levels of cholera toxin conju-
its of filrsrgrsdm (Axons-Olivera at

19920, a trichothecene myco-
toxin (Abouzied et al.. 1993). Although
the mechanism“) by which choleratoxrn
(CT) exerts the potent adjuvant efl’ect in
the immune system is not fully under-
stood. it has.been shown that CTcon-
comrtantly stimulates interleulnn' -1 pro-
duction and antigen presentation (Bro-

FFRR' ’Anv ’oo‘- fnnn V'hl Ode-- -

Immunological Assays (continued)

mander et al.. 1991).
The advantages to this a proach are
severalfold. First, the p urejs rapid

and yield; quality ann'bodieczlm cor;
panson poorer results 'eved
standard tocols. Second. since no
thimpairmentwasobserved
at the concentrationsused inthiswork.
mt be a humane alternative to
. 'sadyumtwhichtypicallygives
rise tooa. ulcers. or granulomas
atthermectionsiteAndthrrd.theuse
ofCT is also valuable when mycotoxin
availability is limited. since relatively

181

uircdto
yre-

low doses of immunogen are
induce a rapid and strong anti
spouse.

Rabbit antisera contain antibodies
generated by multiple B-cell clones.

Thesevarym ’ city andareconsid-
cred yelonal. A moor advantage of
l antisera are that they are of

‘ afinity and inexpensive to pro-
duce. However,inherentvariability£rom
lottolotmakesitdifimlttometbem in
commercial kits with defined perfor-
mancecharacteristica.Hybridomas have
therefore been developed by fusions of

immunized mouse spleen cells with a
myeloma cell line to secrete reagent-
mhéty monoclonal antibodies. A maJo' r
° vantagetothisapproachisthere—
quirement for a tissue-culture facility,
high cost. and time efl'ort involved.

Mycotoxin Immunoassay
Formats

A number of immunoassay formats
have been devised for mycotoxin analy-
sis. Initially, competitive RIAs were used
whereby specific antibody is incubated
with a constant amount of radiolabeled

 

figZ—Preparationotlrmnuiogensbr
wmwmmwloom

 

  

122 $600 rsmocv—sssauanv 1995

.
#4:»! '
o - ' .

 

 

 

 

 

 

Vi

toxininthepresenceofstandardorun—
known sample and then various proce-
duresareusedtoremove the toxinanti-
body complex from solution. The

amount oftoxin in a sample is inversely'

related to the amount of radiolabeled

(imbound) toxin in solution. Because of '

inherent problems with radioactivity.
titive assaysbasedonELISAGEIn-
andPerlman, 1971) were devised.
thdirectand indirectassays (Fig.5)
have been applied to mycotoxin detec-
tion. Microtiter plates. beads. and
Terasaki plates have been used as solid-

182

phase support for ELISA (Pestka et al.,
1980' Pestka and Chu. 1984). High-pro-
tein-binding polystyrene microtiter
plates have been most widely used be-
cause they ofl'er an extensive support
technglzfy, ,including removable stripe.
multi pipettes, automated washers.
and spectrophotometers.
Membranes are an alternative solid
phase that have been employed for

yea-no or threshold tests in cups, cards,
and di 'cks. We have successfully em-
pl nitrocellulose membranes in a

Computer-Assisted-Multianalyte Assay

System (CAMAS) for fum afla-
toxins, and zearalenones (Abouzied and
Pestka, 1994). Monoclonal anti bodies
for each of these toxins are immobilized
as multiple lines on niuocellulose' mem-
brane strips and sectored into hydro-
phobic compartments to minimize use
oiments. A modified ELISA rs wand
whereby tree m
horseradish peroxi beled myco-
toxins compete for binding to them
cellulose-bound aneta'bggiea. Color inten-
sr 0 lines cm a precipitating
tEstate is inversely related to myco-

 

 

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Wmmfimtafl‘flbd—
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gaead and. 9 over Wane-born!
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gate

 

 

 

 

 

 

FEBRUARY 1995—FOOD TEONOLOGY 123

lmmunological Assays (continued) 183

 

 

 

 

 

 

 

Monitor | Z

 

 

 

 

 

 

 

 

 

 

 

 

 

NOW

Antibody lines

 

 

 

 

 

Hg. BMWMMMhWWWWIU
mammmnmslum

1. SEPARATE HAPTENS ON
CHANNELED
HPTLC SILICA GEL PLATE (I)

2. IMMUNOSPECIFIC TRANSFER:

 

 

 

r g FILTER PAPER (F38)
—_ ANflBODYCOA NC_(-)
_ HPTLC (-)

8 8 8' 3. meme

_ WITH HAPTEN-HRP
CONJUGATE (8)

4. INCUBATE NC WITH
PRECIPI‘I'ATING HRP
SUBSTRATE. IDENTIFY
HAPT'ENS AS
INHIBITORY BANDS

 

5. PERFORM SCANNING
DENSITOMETRY

 

 

 

fig. 7—EUSAGRAM Procedure for Mycotoxins. From Pestka (I991)

124 F000 TECHNOLOGY—FEBRUARY 3995

toxin concentration. Line density can be
quantitatively assessed using a camera,
video monitor, and microcomputer
equipped with a video digitizing ' board
(Fig. 6). The assay can be used to deter-
mrne range values for the various myco-
toxinsinexuactsofspikedcominless
than30minandrecordthemonthemi-
comm uter hard disk.

Ano er immunoblot approach, called
ELISAGRAM, has been devised that
combines the sensitivity and selectivity
of competitive ELISA with the capacity
of high-performance thin-layer chroma-
tography (HPTLC) to separate struc-
turally related mycotoxins (Pestka,
1991). The procedure (Fig. 7) involves
separation of mycotoxin by HPTLC,
blotting of the HPTLC plate with nitro-
cellulose (NC) coated with mycotoxin-
specific monoclonal antibody, incuba-
tion of NC with mycotoxin-enzyme con-
‘ugate to identify unreacted antibody

inding sites, detection of bound en-
zyme conjugate with a precipitating sub.
strata, and visual or densitometric as-
sessment of inhibition bands indicative
of a cross-reacting mycotoxin. The tech-
nique has been applied to two major
mymtoxin families, the zearalenones
and aflatoxins. Multiple standard curves
for the zearalenones and the aflatoxins
can also be constructed using scanning
deusitometry. Cross-reactivity in ELIS-
AGRAM curves are analogous to that
found in com titive ELISA. This pro-
cedure could widely applicable to the
simultaneous quantitation and confir-
mation of multiple ha tens with a single
cross-reactive anti ‘ y.

Mycotoxin antibodies have also been

i “figs: 1’ ”I r “d ' l

or or examp e,
aflatoxins can 3% to an afinity
column and then desorbed for subse-
quent derivatization and fluorescence
measurement (Trucksess et aL, 1991).
This approach requires fluorescent my-
cotoxin derivatives and additional in-
strumentation (fluorometer). Another
:Eproach is to quantitate mycotoxin in

e afinity column eluates by
chrolmgnawsnphy (renewed' by u.

Immunoassay of
Mycotoxins in Foods

Antibodies (polyclonal and mono-
clonal) havebeeumadetothemajor
known mnotoxins, and both ELISAs
andRIAs vebeensuceeasfully applied
to the ' of mycotoxins in a
diverse arrayof oodaTable 3 givease~
lected examples of reported immunoaso
says for the aflatoxins, ochratoxins, tri-
chothecenea, fumonisins, and other my-
cotoxins, many of which can detect
picogram or nanogram levels of toxin.
ago mainf gmwu? protein struc-.

0 anti an enzyme co
gate, immunoassays have to be can";
out in an aqueous system. In a liquid
system such as milk, mycotoxins can lie
analyzed directly (Pestka et al., 1981a),
although the limit of detection can be
Increased by cleanup and concentration

—Text continued on page 127

184

 

 

 

Tabla 3—Selocted WWI Assays tor Mycotoxins In Foods

 

 

. " Unit 0'
Toxin Fervent Food analyzed detection Mm
Allatcno‘n a. an Com. wheat. man s nglg EI-Naltb et al. (1981)
butter
ELSA Corn. wheat. pom 3 nglg EI—Nakb at al. (1981)
m
Pesriut butter 2.5 nglg Mortimer et at
(1988)
Corn. cotton-seed 2.5 nglg Dixon at al. (ISBBI
Barisy 0.1 nglml. Ramaluishna at al.
I1990I
Allatoxins B. ELISA Peanut butter 0.3 nglg Morgan at al.
and G; (1986”
Aflatatin M. RIA Mat 0.5 nglg Pestka at d. (1981!»
ELISA Milt 0.3 nglg Pestka et al. (ISBIbl
0.3 nglg Fan et al. (1984)
12 pglg Nieuwenhot at al.
(1990)
AC Milk so pglg Hansen (1990)
Cydopiucnic add ELISA Buffet 30 pg/assay Heron arr! Weilel’
1991
Ergot altaloid ELSA Wheat 10 nglmL Shelby and Kelsy
1992
Fumonisins ELISA Feed 250 nglg Arcana-Olivera at al.
, (19928. bl
firsamchromanone LC-ELISA Wheat. barley 5 only Yu and Chu I199“
Octratoxin ELSA Bartay 1.0 nglml. Rarnaluishne at al.
(1990!
5.0 nglg Candish at al.
I1988I
0.1 nglg Morgan et al.
(1983)
Pig kidney 0.5 nglg Morgan at al.
(1986!)
Wheat. meat. 1.0 nglg Sato at al. (1987)
please
Chicken meat. 0.1 nglml. Kama et al.
wheat flow. (1989. 1990!
porcine plasma.
bovine sera
PR Toxin RIA Grease 50 jag/9 Wei and Chu (1988)
Ribr'atarin RIA Cdtta‘es 0.1 pg Davis and Stone
I197”
Sterigmatocystin ELISA Barley 0.01 pg et al.
“98“
Trichothecenes:
W ELSA Rim 1 nglg Kemp at d. (1985,
valenol
OW ELSA Crltm 16 09/01. M at al. (1989)
scirpencl
Vllhoat 0.3 ug/mL ms at al. "983i
W RIA Corn. wheat 20 nglg Xu et al. IISOGI
ELISA Corn 200 nglrnL Casale et al. (1988l
Wheat 0.1 ng/assay Mile at al. (1990)
W load I pglg Abfuasd at al.
199“
Nivalenol ELISA Barley 0.1 nglassay Itebudti et al. (199“
Roridn A E.SA Feed 5 nglml. W et al.
(1988)
7-2 toxin ma Com. m 0.1 nglg Lea and cm
(1981!)
Mir 2.5 nglg Lee and Our
(1981”
ELSA Com 50 nglg W at d.
(1984)
Com. when 2.5 nglg Pestka et al.
(1981“
.. MI: 0.2 nglg Fan Ct UL (ISM
\Nhoot " 0.5 nglg Chin et al. (1938)
Zearalenone ELISA Contestants“ Inn/ml. LiuetaLI1985l
Corn 1 nglg Warner and Pestka
"986)
We 2.5 nglg Warner and Pestka
toods (1987)

 

 

FEBRUARY 1995—FOOD TECHNOLOGY 125

 

 

 

' Immunological Assays (continued)

185

 

 

 

ru4wmmmmmm~um 1993

 

 

. m Annals “lees-y
tum: mu m who» end- w W m
EZ-Saaen‘ M ELSkWIi- 5-20 10 5.00-1.50 Mm Moses/hi
ahead
M”. WW6- 0.5 10 5.00-7.50 Mir Mons/III
ahead
Ocular-in mm 5 I0 5M7“ Corn Mose-Ital
ahead
1’4 train 8.5K: Mae'- 11.5 IO 5.00-7.50 Corn Vin-I. Mal
ahead
leer-alarm» 85k” 50 10 5.00-1.50 Corn Mose-Ital
ahead
atePrube‘ m5, Whey 5.20 5 10.00 (hum Moan/MW“
“5.10. W ELSkCcp 5.10.20 5 4.00 mumps-nut “WWW
2000' hut-.eenonaeed. “WWW
endIeeda
One-Step M5. ELISA: 5 40 1.00 Commune-an Quantitative: withELSAreeder
EUSA' W Nahum
endIeede
MM. ELSA: 0.5 ‘0 LI» Mi szhhwm
Zeualarlene euse: 100 so I.” Cam.“ Muhammad-r
w
W M am 5 5.10 3.50 Comm _Vaerder&SAree¢er.m
m Mind USOA-EGSaMAOACF‘nt
Actienw
W’ ELSA: 1M 11-20 5.50 CeruwheetJeerl ”www.mflel
Hand m We.“
W
fiancnia‘n 845k 500 12-20 5.50 Market.” thalerELISAreed-Jleaeflel.
M We.“
Ode-train am 20 12-20 5.75 “EM“ Narmada.“
m
74w 85k 500 ".30 5.50 Calumet.” Via-deraISAreaa.oeaeIIel
W
W ELISA: 250 ”-30 5.50 Commune “casement.“
w
Veratu' Anetta-r. EM 5 I5 4.00 Canoe-Ina. Wanna”.
W mm mm
MM. EBA: 0.15 40 5.50 “It WWW“.
W
Der-m6- flJSk :00 10 5.50 Can't-eat.“ WWW“.
“and w
7-2 EBA: 50 30 em Contented Mariam”.
herd-rune m 250 so em Mme.“ wmmnea.
W
Dosage. m5. EBA: 0.5 45 4.00 Canoe-numbed Wanna”
W
W Anetta-rial. White 1.5 45 em “umber! Warned-r.”
.Afleeeet’ Anne-“m Meal- 1.0 to 1.00 Commie“ WWW.
mun WWW“
' WWMACW
Alec-“n". Med- 0.‘ 10 tom MI; Warm
«an
M m Meal- 2 15 tom Cern WWW
.9.) W
W m Meal- 5 10 10.00 Corn WWW
m
M leeralerrerre Med- “ 10 ION Corn.“ WWW
m

 

Whammwm
unmet-WM“

mummznrswmum
fia—mmuzmm
WMRMHGWSIW

Wmmumrzm

"Ila. L!“ m em os-nnoaau
M m MA 0214: was»

 

126 'Fooo TECHNOLOGY—FEBRUARY 1995

 

186

 

on a Sep-Pak or afinity column. Food
samples were 0 ' ’ y extracted with a
solvent stan protocols, evapo-
rated. reconstituted in an aqueous
bone: for assay. However, based on our
initial observation that mycotoxin-
horseradish peroxidase and solid-phase
antibodies retain suficient stability for
ELISA when incubated with as much as
35% (w/vol) methanol (Ram et
1986a). a direct apgsroach was develo
whereby solid su trates are blended
with methanol-water extraction solvent
and the extract analyzed directly or af-
ter dilution.

Immunoassay and various chromato-
graphic methods for mycotoxin detec-
tion in foods are usually comparable
when they are performed in the research
laboratory (Ram et al., 1986a. b; Chu et
al., 1987). However, sometimes toxin-
free food extracts m interftrilre ‘lirtlh
mycotoxin-enzyme ' ingto eso i -
phase antibody and therefore yield a low
falseepositive response when compared
to a standard curvzgrepared in extrac-
tion solvent with b er. Samples can be
diluted more extensively to eliminate
this interference, but this will decrease
sensitivity. Alternatively. interference
can be mmimized by incorporating tox-
in-free sample extracts dunng standard-
curve preparation. Another factor that
must be considered is sample H, which
must sometimes be adjusted, prior to
immunoassay, since antibodyhantigen
binding occurs optimally at neutral pH.

Commercial Mycotoxin
Immunoassays

Theaboveresearchhasledtothede-
velopment of a number of commercial
kits that have been marketed in the
United States for food safety verifica-
tion (Table 4). Commercial immunoas-
say kits have generall performed well in
routme' anatllyeee informed in the labo-
ratory and e 5 d (Kocltsow and Tan-
ner, I990; Azer and Cooper, 1991; Domer
et al., 1993). However, when liar-wits et
al. (1993) recalculated the precision per-
fonnance parameters of collaborative
studies for mycotoxins through 1991.
they found that ELISA had somewhat
poorer precision than thin-layer chro-
ma pby and liquid chromatogra-
phy. us. when adopting a commercial
immunoassay for rapid testing of myco-
tonns.food analysts mustcritically evalo
ustethegeteminlightoftheirspecific
needs an the limitations of the assay
(Table 5). ha ed

organisations ve provul'
leadership in evaluating these tests. Of-
ficial First Action approval has been
given for detection of aflatoxins by
AOAC International (formerly Associa-
tion of Oficial Analytical Chemists) in
various commodities. using microtiter-
well (Park et al.. 1989a. b; Patsy st
1992), cup (Trucksess et al.. 1989).
afinity-column (Trucksess et al., 1991)
tests. The US. Dept. of Agriculture’s
Federal Grain InspectionService (FGIS)
has tested and approved a number of
kits for qualitative screening and quan-
titation of aflatoxins (Table 4).

 

Table 5—Suggested Criteria IorAdop-
o'onolsMyeoroxrn .San-
marizedfiomPssdrslIQOBl
LImltsofdetsctionsndaensitivity
0090
Wmfanpidsaeering

‘ snd/orqusntitation

s 'fi’l

 

Effectiveness of recommended ex-
traction procedure

Sample thrwd'aput

Field stabiity

Inter- and he's-assay reproducbility

 

 

 

To further facilitate kit evaluation.
AOAC International has created the
AOAC W Instlitute with th;
char-gee uatingan certrfymg’ ° rapi
test 'ts used for safety screening of
foods. During summer 1993, this Insti-
tute evaluated aflatoxin test kits in con-
junction with :d Memoranggrm 109g; ndetrl;
standing tn Octo w:
£958 whiéfinT ' P the Test Kit

ormance estmg rogram or test
kits that detect aflatoxins in grain. Upon

successful evaluation using protocols by.

FGIS,testkitssreoertifiedtoclaim

“Performance Tested in Accordance ‘1
with Standards Established by FGIS for ~‘

Test Kits to Detect Aflatoxin Residues

in Grain and Grain Products."‘More re-=

cently. two deoxynivalenol ELISAs have
been similarl certified by FGIS. It is
anticipated t food analysts will be
assisted by similar evaluation studies
that focus on mycotoxins of potential
health and economic significance, such
as deoxynivalenol. the fum0nisins, och-
ratoxins. and zearalenone.

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Updated November 1994 from a paper
gdn tedduring the IFT Toxicology and

ety Evaluation Division posium “Im-
munoassay Applications touchIII
attheAnnual Meetingofthelnstitutsof
{'33: Technologists. Chicago. nLJuly 10—14.

 

—Edited Neil H. Met-melee ' Senior
Associate ' an.

 

....

 

 

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