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This is to certify that the
thesis entitled
COMPARATIVE DETECTION OF FUMONISIN BY HPLC, ELISA AND

IMMUNOCYTOCHEMICAL LOCALIZATION IN FUSARIUM CULTURES; AND
POLYCLONAL ANTIBODY PRODUCTION FOR ERGOSTEROL.

presented by

MARIA VICTORIA TEJADA-SIMON

has been accepted towards fulfillment
of the requirements for

MASIER—_degree in W

Major professor ‘

 

Date 45/44/4994—

o-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

 

 

LIBRARY
Michigan State
Unlverslty

 

 

 

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TO AVOID FINES Mum on or baton dd. duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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MSU IoAn Affirmdm Action/Emu Opportunity Inflation
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COMPARATIVE DETECTION OF FUMONISIN BY HPLC, ELISA, AND
IMMUNOCYTOCHEMICAL LOCALIZATION 1N FUSARIUM CULTURES; AND
POLYCLONAL ANTIBODY PRODUCTION FOR ERGOSTEROL.

By

Maria Victoria Tejada—Simon

A THESIS

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1994

ABSTRACT

COMPARATIVE DETECTION OF FUMONISIN BY HPLC, ELISA, AND
IMMUNOCYTOCHEMICAL LOCALIZATION IN F USARI W CULTURES; AND
POLYCLONAL ANTIBODY PRODUCTION FOR ERGOSTEROL.

By

Maria Victoria Tejada—Simon

Fusarium sp. are frequently found in corn worldwide. Some strains such as
F usarium moniltforme and F usarium proliferatum can elaborate a potent mycotoxin
called fumonisin that is associated with several diseases affecting horses, pigs, rats,
chickens and humans. Although a high performance liquid chromatography (HPLC) has
been the primary method used to detect fumonisins, fumonisin B1 antibodies have recently
been developed and used in enzyme-linked irnmunosorbent assays (ELISAs). The purpose
of this project was to: (1) monitor fumonisin production in Fusarium cultures by two
methods, ELISA and HPLC, and (2) localize the mycotoxin by an enzyme-linked
immunocytochemical technique (ELICT) using the same fiimonisin B1 antibody. When
five different strains of Fusarium species were grown in corn and in a liquid medium for
28 days, there was a positive correlation between fumonisin detection by ELISA and
HPLC. However, ELISA consistently yielded higher amounts when compared with
HPLC. ELICT revealed the presence of large deposits indicative of fiJmonisin or
fiJmonisin-like cross reacting compounds in mycelia, macroconidia and microconidia.

ELICT results qualitatively correlated with HPLC and ELISA.

Ergosterol is the principal sterol in fimgi and an important component of the cell
membranes. This sterol has been previously used to measure fiJngal growth in foods. As a
first step in developing antibodies to this compound, ergosterol was derivatized with
hemisuccinate (Erg-HS) and conjugated to bovine serum albumin (Erg-HS-BSA). Six
white female New Zealand rabbits were immunized by subcutaneous injections of Erg-HS-
BSA conjugate followed by four booster injections. An indirect ELISA was used to assess
titers. Although titers as high as 6400 were detectable, the antibody was not usefirl in
detecting free ergosterol via inhibition in a competitive indirect ELISA. The antibody was
evaluated for applicability in the ELICT for a variety of molds and yeasts but failed to
detect presence of ergosterol in the membranes Of these organisms. Thus, although the
antibodies showed high specificity for Erg-HS-BSA conjugate, they did not react with free

ergosterol.

This thesis is dedicated to my family, specially to my parents, who supported my
decision to study in the States, and also to my fiiend Klaus for his help, concern and

encouragement during the past two years.

iv

ACKNOWLEDGMENTS

I would like to thank Dr. H. Pestka for his direction and support during my
Master's degree program. I would also like to thank the other members of my committee,
Dr. P. Hart and Dr. D. Smith for their suggestions and assistance. Much thanks goes to
Dr. M. Pallmer from Neogen Corporation (Lansing, MI), for his help with some
conjugation procedures, and also to Dr. M. Abouzied for his valuable counsel and

support.

TABLE OF CONTENTS

page
LIST OF TABLES ..................................................................................................... viii
LIST OF FIGURES .................................................................................................... ix
PART I
Abstract .......................................................................................................... 2
Introduction .................................................................................................... 3
Background ......................................................................................... 3
Diseases associated to fumonisins ........................................................ 5
Analytical methods ............................................................................. 6
Rationale ............................................................................................... 9
Material and Methods .................................................................................... 10
Chemicals and reagents ....................................................................... 10
Fungal strains ...................................................................................... 10
Culture conditions .............................................................................. 11
HPLC .................................................................................................. 12
Competitive direct ELISA ................................................................... 13
Enzyme-linked immunocytochemical technique ................................. 14
Protein assay and nitrocellulose membrane blots ................................ 15
Statistics .............................................................................................. 16
Results and discussion .................................................................................... 17
Fumonisin B1 detection by ELISA, HPLC and ELICT
in F usarium cultures .......................................................................... 17
Possible protein-fumonisin association ................................................ 21
Conclusions ..................................................................................................... 21
PART II
Abstract .......................................................................................................... 49
Introduction ................................................................................................... 50
Rationale ................................................................................................. 5 1
Material and Methods .................................................................................... 52
Chemicals and reagents ....................................................................... 52
Preparation of immunogen .................................................................. 52
Ergosterol-HRP conjugation ............................................................... 55
Animal immunization .......................................................................... 55

ELISA ................................................................................................ 56

Enzyme-linked immunocytochemical technique ................................. 57

Results and discussion .................................................................................... 59
Conclusions ....................................................................................................... 61

LIST OF REFERENCES ........................................................................................... 67

vii

Table 1.

Table 2.

Table 3.

LIST OF TABLES
page

ELISA and HPLC correlation for different fumonisin B1 producing
F usarium strains ....................................................................................... 28

Summary of results for ELICT for fumonisin B1 localization in

F usarium corn culture mycelia. Cultures included non

firmonisin B1 producing F .graminearum, fiimonisin Bl

producing Fusart'um strains after 3, 5, 7, l4, 21and

28 d incubation time .................................................................................. 29

Typical absorbance values for erg-HS-B SA antiserum collected afier

fourth boost fi'om a rabbit injected with Freund's adjuvant as determined
by CD-ELISA and CI-ELISA .................................................................... 66

viii

LIST OF FIGURES

page

Figure 1. Structure of fizmonisins: a) (Azcona-Olivera et al., 1992);

b) Fumonisin C1 (Branham, 1993) ............................................................ 4
Figure 2. CD-ELISA for fiJmonisin B1 concentration in liquid medium filtrates

for five different fumonisin Bl producing Fusarium strains ...................... 22
Figure 3. CD-ELISA and HPLC for fumonisin B1 concentration in

F. moniliforme M 5986 corn extracts ....................................................... 23
Figure 4. CD-ELISA and HPLC for fumonisin B1 concentration in

F. mom'ljforme M 5958 corn extracts ....................................................... 24
Figure 5. CD-ELISA and HPLC for fumonisin B1 concentration in

F. monilifonne M 5990 corn extracts ....................................................... 25
Figure 6. CD-ELISA and HPLC for fumonisin Bl concentration in

F. proliferatum M 5956 corn extracts ...................................................... 26
Figure 7. CD-ELISA and HPLC for fumonisin B1 concentration in

F. moniltforme M 5982 corn extracts ....................................................... 27
Figure 8. ELICT of fitmonisin B1 producing F. mom'liforme M 5986

at 3, 5, 7, 14, 21, and 28 d culture time .................................................... 30
Figure 9. ELICT of fiimonisin B1 producing F. moniliforme M 5958

at 3, 5, 7, 14, 21, and 28 d culture time .................................................... 32
Figure 10. ELICT of fiimonisin Bl producing F. monilt'forme M 5990

at 3, 5, 7, 14, 21, and 28 d culture time .................................................... 34

Figure 11. ELICT of fumonisin Bl producing F. proliferatum M 5956
at 3, 5, 7, 14, 21, and 28 (1 culture time .................................................... 36

Figure 12. ELICT of fumonisin B1 producing F. monilifonne M 5982
at 3, 5, 7, 14, 21, and 28 d culture time .................................................... 38

Figure 13. Staining controls for solid corn culture fi'om different
fumonisin B1 producing F usarium strains for ELICT studies ................... 40

Figure 14. ELICT of non fumonisin B1 producing F. graminearum
at 3, 5, 7, 14, 21, and 28 d culture time .................................................... 42

Figure 15. CD-ELISA and HPLC for fumonisin Bl concentration in
F. graminearum, F. proliferatum M 5956 and F. mom'liforme
M 5982 mycelium extracted with acetonitrilezwater (1:1)
from corn cultures ..................................................................................... 44

Figure 16. CD-ELISA and HPLC for fumonisin Bl concentration in
E graminearum, F. proliferatum M 5956 and F. moniltforme
M 5982 mycelium extracted with acetonitrilezwater (1:1)
from liquid medium cultures ..................................................................... 45

Figure 17. ELICT of fumonisin B1 producing Fusarium species in corn
after extraction of mycelia with acetonitrilezwater (1:1) ............................ 46

Figure 18. Structure of A) ergosterol, B) ergosterol" (cyclic Diels
Alder adduct), C) ergosterol*-hemisuccinate derivative,
D) ergosterol‘-hemisuccinate—bovine serum albumin
conjugate, and E) ergosterol-hemisuccinate—BSA (immunogen) ................ 53

Figure 19. ELISA titration of the sera obtained after first boost with Erg-HS-BSA
immunogen for six different rabbits, using Erg-HS-B SA as solid phase ...... 62

Figure 20. ELISA titration of the sera obtained after second boost with Erg-HS-BSA
immunogen for six different rabbits, using Erg-HS-BSA as solid phase ...... 63

Figure 21. ELISA titration of the sera obtained after fourth boost with Erg-HS-B SA
immunogen for six different rabbits, using Erg-HS-B SA as solid phase ....... 64

Figure 22. ELISA titration of the sera obtained after fourth boost with Erg-HS-B SA
immunogen for six different rabbits, using BSA as solid phase .................. 65

PART I

COMPARATIVE DETECTION OF FUMONISIN BY HPLC, ELISA, AND
IMMUNOCYTOCHEMICAL LOCALIZATION 1N F USARI UM CULTURES

ABSTRACT

COMPARATIVE DETECTION OF FUMONISIN BY HPLC, ELISA, AND
IMIVIUNOCYTOCHEMICAL LOCALIZATION IN F USARI W CULTURES

By

Maria Victoria Tejada-Simon

Fusarium sp. are frequently found in corn worldwide. Some strains such as
F usarium moniliforme and F usarium proliferatum can elaborate a potent mycotoxin
called fumonisin that is associated with several diseases affecting horses, pigs, rats,
chickens and humans. Although high performance liquid chromatography (HPLC) has
been the primary method used to detect fumonisins, fumonisin B1 antibodies have recently
been developed and used in enzyme-linked immunosorbent assays (ELISAs). The purpose
of this project was to: (1)monitor fumonisin production in Fusarium cultures by two
methods, ELISA and HPLC, and (2)localize the mycotoxin by an enzyme-linked
immunocytochemical technique (ELICT) using the same fumonisin B1 antibody. When
five different strains of Fusarium species were grown in corn and in a liquid medium for
28 days, there was a positive correlation between fumonisin recoveries by ELISA and
HPLC. However, ELISA consistently yielded higher recoveries when compared with
HPLC. ELICT revealed the presence of large deposits indicative of fumonisin or
fumonisin-like cross reacting compounds in mycelia, macroconidia and microconidia.

ELICT results qualitatively correlated with HPLC and ELISA.

INTRODUCTION

Background. Fumonisins are a group of mycotoxins isolated from Fusarium
species, that include F usarium moniliforme and F. proliferatum (Gelderblom et al.,
1988a). This is one of the most prevalent fungi associated with corn used as human food
and animal feed. Fumonisin B1, the major fitmonisin produced by F. monilt'fonne, is
capable of producing leukoencephalomalacia in horses (Marasas et al., 1988a), pulmonary
edema in pigs (Harrison et al., 1990) and also is a hepatocarcinogen in rats (Gelderblom et
al., 1991). Fumonisin B1 has also been epidemiologically linked to human esophageal
cancer (Marasas et al., 1988b; Yang et al., 1980).

Fumonisin B1 is a C20 aminopolyol with a molecular weight of 721. It is a diester
of tricarballylic acid and polyhydn'c alcohol that is very similar in structure to sphingosine
(Figure l). Sphingosine is the chemical backbone of all sphingolipids, including
sphingomyelin, ceramides and gangliosides. Sphingolipids play critical roles in a number of
cellular functions, including cell-cell communication, growth factor receptors and growth,
differentiation and transformation of cells. Disruption of sphingolipid biosynthesis could
thus have severe consequence for an organism's health. The similarity of fumonisin B1
with sphingosine led to the hypothesis that fumonisins may interfere with sphingosine
metabolism (Wang et al., 1991). Riley et a1. (1993) have documented evidence supporting
this hypothesis. Clearly, more research is needed on how these mycotoxins might
adversely affect animal and humans diseases as well as diagnostic methods to accurately

and sensitively determine fumonisins on food and feed.

 

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5

Diseases associated with fumonisins. Diseases associated with F. moniliforme
and fumonisins include equine leukoencephalomalacia (ELEM), porcine pulmonary edema
(PPE), hepatotoxicity and hepatocarcinogenicity, esophageal cancer (EC) and also
possible effects on the immune system (Norred et al., 1993). Jaskiewicz et al. (1987)
observed liver toxicity when feeding different species with corn contaminated with F
monilr'forme. Liver tumors were produced in rats by feeding F. mom'lr’forme culture
material (Marasas et al., 1984b). Voss et a1. (1989; 1990) investigated mechanisms of this
hepatotoxicity using F. moniliforme culture material or naturally contaminated material.
They established that the causal agent was water-extractable rather than chloroform-
extractable. Gelderblom et al. (1988b) similarly attempted to identify toxic metabolites and
determined that this water-soluble material contained a potential tumor-promoting agent.
By chromatographic procedures, the fumonisins were isolated and their structures
determined (Bezuidenhout et al., 1988; Gelderblom et al., 1988a). Subsequently, extracts
obtained from ELEM cases and hepatotoxicity in rats were examined for the presence of
fumonisins and the compounds were found (Plattner et al., 1990).

ELEM is a neurotoxic disease of horses, mules and donkeys characterized by
liquefactive necrosis of predominantly the white matter in the cerebral hemispheres of the
brain (Marasas et al., 1988a). Outbreaks occur sporadically in many countries. ELEM
typically develops over a several-week period after horses are fed with moldy corn. Then,
horses become lethargic, disoriented and may refuse feed. They become fi'enzied, aimlessly
circling, they exhibit head pressing, convulsions, hyper-excitability and finally death.
ELEM has been produced by feeding horses with com naturally contaminated with F.
monilr‘forme and with inoculated and cultured corn with isolates of this fungus (Buck et
al., 1979). After the discovery of fiJmonisins and their association with ELEM, this disease
was reproduced by administration of purified fumonisin B1 (Marasas et al., 1988a).

In 1989 an outbreak occurred in Georgia where 34 mature swine died with PPE.

The thoracic cavities on those pigs were overfilled with golden-yellow liquid with lung

6

tissue showing severe edema. This epizootic appeared to represent an unrecognized
disease that was perhaps due to an unusual toxin (Harrison et al., 1990); it was
reproduced in younger swine when they were fed with the same corn. F. moniliforme and
F. proliferatum were isolated from the corn (Osweiler et al., 1992) and were found to
produce high levels of fumonisin B1 and B2 (Ross et al., 1990). Relately, PPE and
hydrothorax occurred in a pig that died after receiving four daily intravenous injections of
pure fumonisin B1 (Harrison et al., 1990). Hascheck et al. (1992) suggested a mechanism
for induction of PPE by fumonisin B1. Fumonisin produces toxicity in liver affecting
sphingolipid biosynthesis what leads to damaged membranes from hepatocytes which are
released to bloodstream. Those are phagocytized in lungs, releasing enzymes and other
mediators that increase capillary permeability in the lung tissue, resulting in edema. PPE
has not been found in other species, perhaps because levels of pulmonary macrophages in
other species are much lower (Winkler, 1988).

Correlations between esophageal cancer and F. monilr’forme have been reported in
South Africa (Marasas et al., 1988b) and China (Cheng et al., 1985). Fumonisins have
been implicated as possible causes or contributing factors in this disease. Sydenham et a1.
(1990) performed a study in which moldy and healthy corn samples were taken from high
and low esophageal cancer rate areas of the Transkei. Fumonisin B1 in samples from the
high-rate area was about four times higher than those fi'om the low esophageal cancer
area. That fumonisin B1 is a complete carcinogen has not been established but it appears
to be a potent tumor promoter (Gelderblom et al., 1988b, 1991, 1992). Thus, although
fumonisins may be involved in human esophageal cancer, other possible contributing
factors cannot be excluded.

Analytical methods. Analytical methods for detecting and quantitating firmonisins
used so far are thin layer chromatography (TLC), high performance liquid
chromatography (HPLC), gas chromatography- mass spectrometry (GC-MS), and, more
recently, enzyme linked immunosorbent assay (ELISA). For example, Gelderblom et al.

7

(1988a) used TLC on reverse-phase plates. Developing solvent was methanolzwater (3:1)
and 0.5% p—anisaldehyde and heating (120°C)were used to visualize fumonisins. While
qualitatively usefirl, this and other methods are not accurate for quantifying firmonisin
levels.

An HPLC method was developed for fumonisin quantitation by Gelderblom et al.
(1988a). HPLC of firmonisins is possible by analysis of its maleyl derivative (ultraviolet
detection at 230 nm). The maleyl derivatives elute fi'om a reverse-phase C13 column using
methanolzo. 1M potassium phosphate buffer (pH 3.8)(7 :3). A more sensitive HPLC
method to determine firmonisins (Bland B2) has been reported by Shephard et al. (1990).
This is based on methanolrwater extraction, anion exchange cleanup and isocratric,
reverse-phase chromatography using fluorescence detection of the preformed o-
phthaldialdehyde (OPA) derivatives. Detection limits for this method are 50-100 ng/g.
Sydenham et al. (1992) recently modified Shephard's method to include fumonisin B3.
Another liquid chromatographic method was also described by Stack et al. (1992). Here,
after extraction of the fumonisins with methanolzwater (3:1), clean-up with strong anion
exchange column, elution from the column with methanolzacetic acid (199 + 1) and
evaporation of eluate to dryness, the metabolites are derivatized with CPA and 2-
mercaptoethanol to make a fluorescent derivative. Reverse-phase liquid chromatography
with fluorescence detection is used for final determination using acetonitrile:water:acetic
acid (50+50+1) as the mobile phase.

One disadvantage of the above described fluorescence derivatizing reagents
currently used for liquid chromatography of firmonisins are instability of the derivatives.
Scott et al. (1992) described a liquid chromatographic method using 4-fluoro-7-
nitrobenzofirrazan (NBD-F), in where the derivatives formed are moderately stable and the
range of detection is l ng/g of fumonisin B1 and B2. However, derivatization with NBD-F
required many steps and was time consuming. Presence and quantification of fumonisins

can also be done by using gas chromatography-mass spectrometry (GC-MS). This is a

8

sensitive and selective method but the disadvantage is that the equipment is very expensive
(Plattner et al., 1990).

Recently, Azcona-Olivera et al. (1992a,b) developed polyclonal and monoclonal
antibodies to fumonisin B1, fumonisin B2 and fumonisin B3; these were used in rapid
competitive enzyme-linked immunosorbent assays (ELISAs) determining firmonisin levels
in feed. Fumonisin was conjugated at the amino group position. Polyclonal antibodies
were produced after immunization of mice with firmonisin Bl-cholera toxin conjugate
without adjuvant. These antibodies cross-reacted with firmonisins B2 and B3 but not with
the hydrolyzed backbone of fumonisin B1 and tricarballylic acid (Azcona-Olivera et al.
1992b). This suggests that the immunodominant portion of fumonisin conjugate is near the
union of the tricarballylic acids to the GM to C-20 positions of the molecule. Monoclonal
antibodies were also prepared from hybridomas selected after fusing splenic lymphocytes
fi'om mice immunized with firmonisin Bl-cholera toxin conjugate with NS-l myeloma
cells. Again, antibodies cross-reacted with firmonisins B2 and B3 (Azcona-Olivera et al.
1992a). When applied to the direct ELISA, both polyclonal and monoclonal antibodies,
exhibited excellent recovery values on spiking studies. Monoclonal antibodies for
fumonisin B1 (and also for aflatoxin B1 and zearalenone) were also used in a line
irnmunoblot assay for screening of firmonisin B1, aflatoxin B1, and zearalenone
simultaneously using a computer-assisted multianalyte assay system (Abouzied et al.,
1994)

Recently, competitive direct ELISA has been used to monitor firmonisin in food
samples from retail stores and the results compared to GC-MS and HPLC (Pestka et al.,
1994). The results showed correlation between ELISA and the other two methods but
higher estimates by ELISA than by GC-MS and HPLC. Dr. Sydenham (in personal
communication, 1993) also observed higher results for fumonisin levels by ELISA than by

HPLC, on both crude and purified extracts using one strain of F. monilifonne, MRC 826.

9

Rationale. HPLC has been the main method used to detect firmonisins but
requires laborious extractions, cleanups, elutions and finally detection in samples. Afier
fumonisin B1 antibodies were developed, ELISA has been used to screen firmonisins in
food. The purpose of this project was to: (1) compare fumonisin production in Fusarium
cultures from different strains of this fumonisin producing fungus by ELISA and HPLC,
(2) localize the fumonisin in the fungus by an enzyme-linked immunocytochemical
technique (ELICT) using the same firmonisin B1 antibody than for ELISA, and (3)
determine if there is any correlation among ELISA, HPLC and ELICT regarding detection
of fumonisin. Extraction with acetonitrile:water (1:1) (for corn cultures) or a simple
filtration (for liquid cultures), was the only step performed before quantitation of
firmonisins, either by ELISA or by HPLC. This eliminated possible variability of results
due to the diversity of extraction procedures. The results suggested that these three
techniques are correlated, even though ELISA consistently yielded higher recoveries when

compared with HPLC.

10

MATERIAL AND METHODS

Chemicals and reagents. All inorganic chemicals and organic solvents were
reagent grade or better. Calcium carbonate was purchased from Fisher Scientific Co. (Fair
Lawn, NJ); carboxymethyl cellulose fi'om Du Pont De Nemours (Wilmington, DE);
glucose, potassium phosphate monobasic, acetonitrile, methanol, sodium dihydrogen
phosphate, di-sodium tetra-borate, sodium carbonate, sodium bicarbonate, sodium
phosphate, Tris base, ethanol from Baker Chemical Co. (Phillipsburg, NJ); ammonium
sulfate, ammonium nitrate, magnesium sulfate heptahydrate fi'om CCI (Columbus, WI);
manganese sulfate monohydrate was purchased from Mallinckrodt (St. Louis, MO);
thiamine, riboflavin, pantothenate, niacin, pyridoxamine, folic acid, biotin, vitamin B12, 0-
phthaldialdehyde, 2-mercaptoethanol, Tween 20, ovalbumin (grade HI), para-
forrnaldehyde, anti-mouse IgG—HRP, bovine serum albumin (fraction V biotechnology
grade BSA), diaminobenzidine, peroxide, dioctyl sodium sulfosuccinate, 3, 5, 3‘, 5'
tetramethylbenzidine fiom Sigma Chemical Co. (St. Louis, MO); ortho-phosphoric acid,
picric acid from Aldrich Chemical Co. (Milwaukee, WI); agar, yeast extract from Difco
laboratories (Detroit, MI); V-8 juice fi'om Campbell (Camden, NJ).

Fungal strains. Isolates from Fusarium species were obtained from the Culture
collection of the F usarium Research Center in Pennsylvania State University (University
Park, PA.) The five strains used included: M 5986 F. monilifonne, isolated fi'om samples
that produced PPE; M 5958 F. monihfonne, isolated from samples that produced ELEM;
M 5990 F. moniliforme, isolated fiom samples that produced PPE; M 5956 F.
proliferatum, isolated fi'om "non-problem” samples; M 5982 F. monilr‘forme, isolated fiom
samples that produced PPE (Ross et al., 1990). In addition F. graminearum W-8, which
does not produce fumonisins, was used as a negative control. This strain was isolated from
scabbed wheat in Michigan during 1981 (Hart et al., 1982). It produces deoxynivalenol

(vomitoxin) and zearalenone (Hart et al., 1982).

11

Culture conditions. Petri dishes with V-8 juice agar medium (Stevens, 1974)
were inoculated with the lyophilized cultures from the Fusarium Research Center Culture
Collection. Each liter of V-8 juice agar medium contained 200 ml V-8 juice, 3.0 g calcium
carbonate, and 20 g agar (Jackson et al ., 1990). These plates were incubated for 10-14 d
at room temperature on an alternating light-dark schedule. The same procedure was
followed for F. graminearum W-8.

To prepare spore suspensions for F. proliferatum and F. moniliforme a loopfull of
conidia from Petri dishes was transferred to V-8 juice agar slant-tubes and these were
incubated at room temperature for 10-14 (1 on an alternating light-dark schedule. To
inoculate ground corn, slants were washed with sterile distilled water to produce conidial
suspensions. To inoculate liquid medium, slants were washed with the same liquid medium
to produce conidial suspension.

F. gramineamm W-8 was transferred to Erlenmeyer flasks (150 ml) 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, l g of yeast
extract in 1 L of distilled water), and then incubated at 25°C under shaking at 220 rpm for
5 d. Cultures were checked every 2-3 (1 for macroconidia production. Conidial
suspensions for F. graminecmmr W-8 were obtained from CMC medium, by removing
mycelia by filtration through 4 layers of sterile cheesecloth.

For corn samples, Erlenmeyer flasks (250 ml) were filled with 40 g of ground corn
and 11 ml of distilled water, and autoclaved for 30 min. After autoclaving, an additional
11 ml of sterile distilled water was added with 107 conidia. The flask cultures were then
incubated in the dark at 25°C for 3, 5, 7, 14, 21, and 28 d. This experiment was carried
out in duplicate.

Liquid medium consisted of glucose 90g/L, ammonium sulfate 3.5g/L, potassium
phosphate monobasic 2.0g/L, magnesium sulfate heptahydrate 0.3 g/L, manganese sulfate

monohydrate l6mg/L, 500 ug/L each of thiamine, riboflavin, pantothenate, niacin,

12

pyridoxamine, 50 ug/L each of folic acid, biotin and vitamin B12 (Jackson et al., 1990).
Glucose was autoclaved separately from salts; vitamin stock solutions (10mg/ 100ml) were
filter sterilized through 0.2 pm filters (Nalgene Co., NY). Initial pH was 5.0. Duplicate
erlenmeyer flasks (125 ml) were filled with 25 ml of this sterile liquid medium and
inoculated with 107 conidia (conidia suspension made in the same medium). Flasks were
incubated in the dark at 25°C2t2°C under shaking at 220 rpm for 3, 5, 7, 14, 21, and 28 d.

Corn cultures were extracted with 5 ml/g of acetonitrile:water (1 : l) by soaking for
2-3 h with mixing every half hour (Plattner et al., 1992). Suspensions were filtered
through Whatman # 1 filter paper (Whatman Ltd., Maidstone, UK). Mycelia were
removed {Tom the liquid cultures by filtration through Whatman # 1 filter paper.

HPLC. The method used for firmonisin B1 determination by HPLC was a
modification of a previously described method (Shephard et al., 1990; Sydenham et al.,
1992). The liquid chromatograhy system consisted of an Isco Model 2300 HPLC pump
with an injector valve (V alco valve) (Lincoln, NE); Hewlett Packard model HP 3392A
integrator (Avondale, PA); H-S3 C18 # 316 stainless steel packed C18 column (Perkin
Elmer, Norwalk, Co) 0258-0178 reverse phase (3.3 cm); Linear Instruments Fluor
fluorescence detector L.C. 304 (Reno, NE) fitted with a 3.1 ul 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 consisted of methanol-0.1M sodium dihydrogen
phosphate (15.6g NaI-I2 P04 . 2 H20 in 1 liter distilled water) (66:34) that was adjusted to
pH 3.4 with ortho-phosphoric acid (PO4H3) and filtered through a 0.45 um Waters HV
membrane (Millipore Corporation, Bedford, MA). The mobile phase was pumped at a
flow rate of 1.5 ml/min.

For preparation of OPA derivatizing reagent, 40 mg o-phthaldialdehyde (OPA)
were dissolved in 1 ml methanol and diluted with 5 ml 0.1M di-sodium tetra borate (3.8 g
in 100 ml distilled water) and 50111 2-mercaptoethanol. The solution was stored for no

more than one week at room temperature in the dark.

13

Standard solutions of firmonisin B1 (provided by Dr. R. Plattner, U.S.Department
of Agriculture, Peoria IL) were prepared in acetonitrile:water (1:1) to give concentrations
fiom 0 to 50 ng/ul. Fumonisin Bl standard solutions (25 ul) were transferred to the base
of a small test tube and mixed with 225 pl of OPA reagent. Because of the instability of
OPA derivatives, it was necessary to prepare the derivatives immediately prior to
injection, and to inject (10 ul) within 2 min of mixing the reagents. OPA forms highly
fluorescent products upon reaction with primary amines in the presence of 2-
mercaptoethanol (Benson et al., 1975). Samples were treated in the same way, taking 501,11
sample, adding 200 111 OPA reagent and injecting 10 ul of the derivative OPA reagent.
Fumonisin Bl concentration was determined by plotting concentration versus peak area.

Competitive direct enzyme-linked immunosorbent assay (CD-ELISA).
Microtiter plates with 96 wells (Immunolon 4 removawell strips, Dynatech Laboratories,
Chantilly, VA) were coated overnight by air-drying at 40°C, with 125 pl firmonisin B1
monoclonal antibodies from ascites (Azcona-Olivera et al., 1992a) (diluted 1:1000) in
0.1M sodium carbonate-bicarbonate buffer (pH 9.6). After washing four times with 300 111
0.02% (v/v) Tween 20 in Phosphate buffered saline (0.01M sodium phosphate/L buffer
containing 0.15M NaCl)(PBS-Tween), wells were blocked for 30 min at 37°C with 300 pl
1% (w/v) ovalbumin (grade III) in PBS (OA-PBS) and then washed four times with PBS-
Tween. Solid samples extracts were adjusted to 12.5% (v/v) acetonitrile:water, and liquid
culture samples were adjusted to pH 7 with PBS prior to ELISA. Mycotoxin standard (in
12.5 % acetonitrile:water for solid culture extracts and in PBS for liquid culture filtrates)
or sample and fumonisin-HRP conjugate prepared by the periodate method (Nakane et al.,
1974) (diluted 1:750 in OA-PBS) were added in 50 ul aliquots consecutively to each well.
After 1 h of incubation at 37°C, plates were washed and bound peroxidase was
determined with 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS substrate)
(Pestka et al., 1982). Fumonisin B1 concentration was determined from standard curves

relating absorbance to firmonisin B1 standard.

14

Enzyme-linked immunocytochemical technique (ELICT). The method of
Lawellin et al. (1977) was used for immunocytochemical localization of firmonisin B1 in
cultures. Fixative solution was composed of 20 g para-formaldehyde to which 150 ml of a
double-filtered, saturated aqueous solution of picric acid was added. Dissociation of the
para-formaldehyde into formaldehyde in the strong picric acid solution was obtained by
heating (60°C) and on alkalinization of the mixture with drops of 0.6N sodium hydroxide
in water. A clear solution was obtained, which was filtered, allowed to cool, and then
made up to one liter with a phosphate buffer (3.31 g NaH2P04~H20, 33.77 g NaZI-IPO4-
7H20, one liter distilled water, pH 7.3). The fixative could be prepared in large quantities
because it is very stable and withstands exposure to light at room temperature for 12
months without deteriorating.

Samples were fixed in phosphate-bufi‘ered picric acid-formalin by treating mycelia
(50 ul) with fixative (1001,11) in an Eppendorf tube for 8 h at room temperature. Sample
tubes were spun at 12,000 rpm in a clinical centrifuge and excess fixative removed. Tubes
were washed twice with 0.01M PBS for 30 min at room temperature, centrifuged again
and excess PBS removed. Fumonisin B1 antibody was diluted 1:50 with 1%(w/v) BSA in
PBS (PBS-BSA) and 100 pl were added to the mycelia. A control was made without
antibody. Samples were incubated for 24 h at room temperature. Vials were spun and
supernatant was removed. Mycelial pellets were washed with PBS-BSA twice by
incubating for 30 min at room temperature followed by centrifiigation. Afier supernatant
was removed, 100 pl of anti-mouse IgG-HRP conjugate (diluted 1:50 in PBS-BSA) was
added to each vial, and incubated for 3 h at room temperature. Samples were washed with
PBS-BSA and, after centrifuging, mycelia were spread onto slides. One drop of this
diaminobenzidine solution (diaminobenzidine 0.005% in 0.05M TRIS [3.023 TRIS BASE,
500 ml distilled water and pH 7.6], pH 7.6 with 0.001% H202) was added and slides were
covered with microscope cover glass. Preparations were sealed with nail-polish and

observed by light microscopy (Nikon brightfield and fluorescence microscope with camera

15

system AFM auto-microflex attachment and Polaroid M-lOO camera, Nikon Inc., Garden
City, NY and Polaroid 667 film, Polaroid Corporation, Cambridge, MA). Slides were
evaluated for brown precipitate indicative of peroxidase staining in a semiquantitative
fashion fi'om 0 to 3+, in which 0 means absent or negative, 1+ slightly positive, 2+
moderately positive and 3+ markedly positive.

Protein assay and nitrocellulose membrane blots. Protein determination on
mycelia] extracts was made in accordance with the method of Bradford (1976) using
commercially prepared dye reagent and standard bovine gamma globulin were purchased
from Bio-Rad Laboratories.

For membrane blots, a template of 1 x 1 cm squares was drawn using a
hydrophobic pen (PAP Research Products International Corp., Mt. Prospect, IL) on a
nitrocellulose membrane (0.45 pm pore size fi'om Spectrum Medical Industries, Los
Angeles, CA). Samples were applied to the membrane template (approximately 1 1.11 per
square) and dried. The nitrocellulose membrane was placed over 2 sheet of Whatman # 1
filter paper to facilitate uptake and drying of samples. The membrane compartments were
blocked with 100 pl of 3%(w/v) OA-PBS for 10 min (to block nonspecific binding sites),
washed three times with PBS-Tween for l min and dried between sheets of Whatman # 1
filter paper. Antibody (diluted 1:100) was prepared in OA-PBS and 1 111 per square was
applied. Membrane was incubated for 10 min, washed three times with PB S-Tween, for 1
or 2 min. Anti-mouse IgG-HRP (diluted 1:100, 1:300, 1:500, 121000, 1:2000 and 1:3000
in OA-PBS) (1 111 per square) was applied and incubated for 15 min, then washed three
times with PBS-Tween for 1-2 min. Fresh substrate was prepared that consisted of 10 ml
of solution consisting on 80 mg dioctylsodiumsulfosuccinate, 24 mg 3,5,3',5'
tetramethylbenzidine, 10 ml ethanol at 56°C (DONS/TMB), 30 ml citrate phosphate buffer
pH 5.0 [kept at 4°C], and 20 pl 30 % H202). Substrate was added to membrane and
incubated at room temperature for 10-30 min. Color was allowed to develop and the

reaction was stopped by rinsing with distilled water.

16

Statistics. For ELISA and HPLC analysis, all values represent means and standard
errors of the means (SEM) of four replicates (n=4). Pearson product-moment correlation
coefficient (r) was used to correlate ELISA and HPLC data. A correlation coefficient
close to 1 indicates there is a perfect positive relationship between the two variables, with
both always increasing together. A correlation coefficient r close to 0 indicates no
relationship between the two variables. A correlation coefficient r close to -1 indicates a
strong negative correlation. The P value is the probability of being wrong in concluding
that there is a true association between the variables. The smaller the P value, the greater
the probability that the variables are correlated (Romano, 1977). Sigma Stat was used to

calculate these correlation coefficients (Jandel Scientific, San Rafael, CA).

1 7
RESULTS AND DISCUSSION

Fumonisin B1 detection by ELISA, HPLC and ELICT in Fusarium cultures.
Fusarium mycotoxin analysis was performed by ELISA, HPLC and ELICT. Liquid
medium cultures and ground corn were incubated for 3, 5, 7, 14, 21, and 28 d. Corn
cultures were extracted with acetonitrile:water (1: 1) and the extracts were analyzed by
ELISA and HPLC. Liquid medium cultures were analyzed only by ELISA because a black
precipitate was produced when the samples were mixed with OPA. This did not allow
injection of those samples into HPLC. The ELISA results showed that fumonisin B1 was
produced in liquid media (Figure 2) in lower quantities than in solid media (Figures 3 to
7). Fusarium cultures grown in defined liquid medium produced detectable levels of
firmonisin B1 after 3 d for the strains M 5958 and M 5982 (3.2ug/ml) even though not
apparent in Figure 2 because the scale used. The highest levels were detected at 28 d
incubation time except for M 5982 (at 14 d). The results suggest that ELISA may be
suitable for screening of firmonisin B1 production by toxigenic strains of Fusarium species
in liquid culture.

Analyses of ground corn cultures are presented in Figures 3 to 7 and reflect
fumonisin B1 levels recorded by ELISA together with the HPLC results obtained fi'om the
same sample. Those results clearly indicate differences in levels detected between the two
methods for all cultures. Controls (i.e. non inoculated samples) analyzed by ELISA
showed very low levels of fumonisin B1 (60 ppm), possibly due to natural contamination.
However, no fumonisin B1 was detected in these control samples using HPLC. Samples
inoculated with non producing fumonisin B1 F. graminearum (negative controls) also
yielded a positive response by ELISA (50 ppm), but did not increase with culture time.
Fumonisin B1 was undetectable in F. graminearum by HPLC. The concentration of
fumonisin B1 detected was insignificant when compared with F. monilt'forme and

F. proliferatum.

18

In Fusarium corn extracts, both ELISA and HPLC techniques detected increasing
fumonisin B1 production with increasing incubation time up to 14 d. In some cultures, a
sudden decrease at 21 d was observed followed by an increase at 28 d. Exceptions can be
seen for the strains M 5986 (Figure 3) and M 5956 (Figure 6) which show a steady
increase as incubation time progressed. Fumonisin B1 was detected by ELISA in cultures
after 3 d of growth in ground com (800 to 1000 ppm), and after 5 d by HPLC in the same
samples (20 to 130 ppm) at considerable levels, significant respect controls (not apparent
in Figures 3 to 7 because of the scale used). The Pearson product-moment correlation
coefficient was used to correlate ELISA and HPLC data (Table 1). A positive correlation
coefficient was obtained between ELISA and HPLC (Table l). The strains M 5986, M
5990, M 5956 yielded a significant P value (P < 0.05) (Figures 3, 5 and 6). Similar
tendencies were found for strains M 5958 and M 5982, but correlations (r=0.085 and
0.076, respectively) were not significant (P >0.05) (Figures 4 and 7).

Thus, although results suggest that ELISA can be effective as a screening method
for fumonisin in cultures, they showed significantly higher fumonisin levels in ground corn
extracts as compared to those obtained by HPLC. The elevated estimates for fumonisin B1
in cultures by ELISA compared to HPLC have been also found in food (Pestka et al.,
1994), but the difference (up to 30-fold in food) was much higher in cultures (up to 400-
fold). This disagreement may be related to several possibilities. First, these differences may
result fiom the presence of compounds with similar structures in extracts which are
detectable by ELISA but not by HPLC. The antibody may not be totally specific for
known firmonisins, but can possibly detect other related compounds. Secondly, due to the
fumonisin stmcture, this mycotoxin might form micelles, such structures may enhance
inhibition in the competitive assay and thus increasing the mycotoxin detection by ELISA
without affecting the HPLC detection. A third possibility is that firmonisin producing

Fusarium species produce fumonisin-conjugated compounds which might cross react and

19
thus increase the response of the ELISA. These same compounds might not be detectable
by HPLC and therefore account for the differences in both methods.

Visualization of firmonisin deposits within the different strains of Fusarium hyphae
was made possible by using ELICT. Diaminobenzidine was used as a reagent to visualize
the firmonisin-antibody complex. The enzymatically oxidized substrate precipitated,
marking putative firmonisin deposits as dark-brown granules, as could be seen in the five
firmonisin producing Fusarium strains used for this study. Mycelia from liquid medium
filtrates developed a brown color when exposed to the atmosphere or at contact with the
fixative. Since this did not allow an unambiguous determination of the dark-brown
granules inside the mycelia, it was not possible to determine whether those granules were
firmonisin deposits or color acquired for the mycelia. Because of that, only results for
ground corn Fusarium cultures are presented (Table 2, Figures 8 to 14). For all five
different firmonisin B1 producing Fusarium cultures, dark-brown granules indicative of
firmonisin presence were observed inside hyphae, conidiophores and conidia (Figures 8 to
12). However, hyphae and conidiophores were the structures in which fumonisin is
predominantly present; a low percentage of this mycotoxin was visible inside conidia.
Representative controls for different samples are shown in Figure 13. Controls included:
(1) samples stained without incubation with primary antibody (fumonisin B1 antibody), (2)
samples stained without incubation with secondary antibody (anti-mouse IgG-HRP), (3)
non-firmonisin-producing F. gramineamm mycelium (negative control). Different
structures with no fumonisin B1 deposits were evident by absence of dark-brown granules
within either microconidia (spores formed for asexual reproduction on hyphae or on
modified hyphal branches called microconidiophores), microconidiophores or mycelia in
general. The same results were observed for F. graminearum controls on macroconidia
(the larger conidia, multicelled), conidiophores and mycelia (Figure 14).

. Samples of mycelium from three strains were taken and treated with

acetonitrile:water (1:1) to extract internal firmonisin to (I) verify that the fumonisin was

20

stained and (2) the material is extractable. Acetonitrilezwater extracts fi'om mycelia, fi'om
either ground corn or liquid medium cultures, were analyzed by ELISA, by HPLC
(Figures 15 and 16) and acetonitrile:water extracted mycelia were then stained by ELICT
(Figure 17). Treatment did not produce the change of color of the mycelia from those
liquid cultures. This experiment was only performed for E graminearum, F. proliferatum
M 5956 and F. monilifonne M 5982. Extracts of these mycelia analyzed by ELISA and
HPLC yielded similar results to those described above with ELISA results being
significantly higher than HPLC results (Figures 15 and 16). When ELICT was performed
on mycelia extracted with acetonitrile:water (1:1) prior to staining, no fumonisin was
observed. This indicates fumonisin was in an extractable form (Figure 17).

Thus, it was possible to monitor fumonisin in the mycelia, spores and
conidiophores using ELICT. This technique appeared to be a good method for detecting
fumonisin inside mycelia, even though only qualitative statements regarding the fumonisin
production can be made. However, when observing fumonisin production with time,
reliable statements in terms of increase and/or decrease are possible. Thus this technique is
a valuable supplementation to methods like ELISA and HPLC. ELICT with peroxidase-
labeled antiimmunoglobulin permits the gross visualization of intracellular firmonisin,
although it is incapable at the light microscope level of demonstrating the specific site of
firmonisin interaction with cellular components. The data from ELISA, HPLC and ELICT
are in agreement regarding fumonisin production, but the long incubation time needed for
ELICT suggests that this method is not as useful for diagnosticians to rapidly detect
fumonisin B1 in food samples. Nevertheless, ELICT results qualitatively correlated with
HPLC and ELISA. Even though the regular procedure described by Lawellin et al. (1977)
suggests long incubation times (8 h for fixation, 24 h for primary antibody, and 3 h for
secondary antibody), it would be interesting to compare results using incubation times less

than the ones recommended; nevertheless, this method, requires three different

21
incubations, and would always be longer to perform than ELISA or HPLC. Besides the
presence of mycelia is required.

Possible protein-fumonisin association. A protein assay was performed on
acetonitrile:water (1 : 1) corn extracts afier 28 d incubation time (i.e. for a good firmonisin
production). The protein concentration in those extracts was 70 ug/ml for uninoculated
corn control and in a range of 100-200 ug/ml for Fusarium extracts. Nitrocellulose
membranes were used to retain protein from extracts. Pure firmonisin B. standards
(controls) were made by adding solutions containing from 0 to 1000 ng/ml firmonisin B1.
Dot blots were performed to determine if fumonisin was associated with protein. Dot blots
performed did not show any difference between controls and extracts, thus visual
comparison could not be made between controls and samples afier exposing them to
fumonisin B1 antibody. Affinity for firmonisin presence on those extracts was not detected
using firmonisin B1 antibody. This suggested that macromolecular fumonisin was not

present in fractions of the acetonitrile:water extracts.
CONCLUSIONS

The results regarding determination of fumonisin concentration showed differences
between ELISA and HPLC in fumonisin producing Fusarium cultures. ELISA results
were significantly higher than those obtained by HPLC (up to 400-fold in some cultures).
However, ELISA and HPLC results were correlated. Fumonisin was also detected by
ELICT in mycelia, conidia and conidiophores, showing higher tendency to appear in
hyphae and conidiophores than in conidia. ELICT results were also qualitatively correlated

with HPLC and ELISA.

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TABLE l.- ELISA and HPLC correlation for different fumonisin Bl
producing F usarium strains.

r P
smms
M 5986 0.987 0.002
M 5958 0.752 0.085
M 5990 0.856 0.029
M 5956 0.881 0.020

M 5982 0.766 0.076

29

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30

FIGURE 8.- ELICT of fumonisin B1 producing F. moniliforme M 5986 at 3, 5, 7, 14,
21, and 28 days culture time. Note the dark granules within the hyphae representing the
fumonisin deposits. 3) 3 days incubation time. Septate hyphae (x 40). b) 5 days incubation
time. Microconidia (x 40). c) 7 days incubation time. Septate hyphae (x 100). d) 14 days
incubation time. Arrow (1) indicates macroconidiophores, arrow (2) indicates hyphae (x
100). e) 21 days incubation time. Septate hyphae (x 40).

 

32

FIGURE 9.- ELICT of fumonisin B1 producing F. moniliforme M 5958 at 3, 5, 7, 14,
21, and 28 days culture time. Note the dark granules within the hyphae representing the
fumonisin deposits. a) 3 days incubation time. Arrow indicates microconidia (x 100). b) 5
days incubation time. Arrow ( 1) indicates microconidia produced in aerial mycelium, arrow
(2) indicates microconidiophores (x 40). c) 7 days incubation time. Arrow (1) indicates
microconidia, arrow (2) indicates mycelium (x 100). d) 14 days incubation time. Mycelia (x
40). e) 21 days incubation time. Septate hyphae ( x 100). i) 28 days incubation time.
Arrow ( 1) indicates mycelia, arrow (2) indicates microconidiophores (x 40).

110
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34

FIGURE 10.- ELICT of fumonisin Bl producing F. monilrjorme M 5990 at 3, 5, 7, 14,
21, and 28 days culture time. Note the dark granules within the hyphae representing the
fumonisin deposits. a) 3 days incubation time. Arrow (1) indicates microconidiophores,
arrow (2) indicates septate hyphae (x 40). b) 5 days incubation time. Microconidia (x 40).
c) 7 days incubation time. Hyphae (x 100). d) 14 days incubation time. Mycelia (x 40). e)
21 days incubation time. Mycelia (x 100). t) 28 days incubation time. Mycelia (x 100).

 

36

FIGURE ll.- ELICT of fiJmonisin B1 producing F. proliferatum M 5956 at 3, 5, 7, 14,
21, and 28 days culture time. Note the dark granules within the hyphae representing the
fumonisin deposits. 3) 3 days incubation time. Microconidia (x 40). b) 5 days incubation
time. Microconidia ( x 40). c) 7 days incubation time. Arrow (1) indicates
microconidiophores, arrow (2) indicates septate hyphae (x 40). d) 14 days incubation time.
Hyphae (x 100). e) 21 days incubation time. Arrow (1) indicates microconidiophores,
arrow (2) indicates hyphae (x 40). t) 28 days incubation time. Mycelia ( x 100).

 

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38

FIGURE 12.- ELICT of fumonisin B1 producing F. monilrforme M 5982 at 3, 5, 7, 14,
21, and 28 days culture time. Note the dark granules within the hyphae representing the
fumonisin deposits. a) 3 days incubation time. Arrow indicates microconidiophores (x 40).
b) 5 days incubation time. Mycelia (x 40). c) 7 days incubation time. Hyphae (x 100). d)
14 days incubation time. Arrow (1) indicates microconidiophores, arrow (2) indicates
septate hyphae, arrow (3) indicates microconidia (x 40). e) 21 days incubation time. Arrow
(1) indicates microconidia, arrow (2) indicates hyphae (x 100). t) 28 days incubation time.
Macroconidiophores (x 40).

 

40

FIGURE 13.- Staining controls for solid corn culture from different fumonisin Bl
producing Fusarium strains for ELICT studies: a) F. proliferatum M 5956, 3 days
incubation time, without fumonisin B1 antibody. Microconidia (x40). b) F. proliferatum
M 5956, 5 days incubation time, without anti-mouse Ig G-I-mP. Microconidia (x40). c) F.
moniliforme M 5958, 7 days incubation time, without fizmonisin B1 antibody. Arrow
indicates microconidiophores (x40). d) F. proliferatum M 5956, 14 days incubation time,
without fiimonisin B1 antibody. Microconidia produced in the aerial mycelium (x40). e) F
monilifonne M 5990, 21 days incubation time, without anti-mouse Ig G—HRP. Septate
hyphae (x 40). t) F. monilzfonne M 5990, 28 days incubation time, without anti-mouse Ig
G-HRP. Mycelia (x 100).

 

42

FIGURE l4.- ELICT of non fiJmonisin 31 producing F. graminearum at 3, S, 7, 14, 21,
and 28 days culture time. a) 3 days incubation time. Arrow indicates conidiophores (x40).
b) 5 days incubation time. Mycelia (x40). c) 7 days incubation time. Mycelia (x 40). d) 14
days incubation time. Mycelia (x 100). e) 21 days incubation time. Macroconidia (x 100).
f) 28 days incubation time. Arrow indicates macroconidia (x 40).

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FIGURE l7.- ELICT of fumonisin B1 producing Fusarium species in corn after
extraction of mycelia with acetonitrile:water (1:1). a) F. proliferatum M 5956, 28 days
incubation time. Arrow (1) indicates macroconidia, arrow (2) indicates microconidia (x
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PART II

POLYCLONAL ANTIBODY PRODUCTION FOR ERGOSTEROL

48

ABSTRACT
POLYCLONAL ANTIBODY PRODUCTION FOR ERGOSTEROL
By

Maria Victoria Tejada-Simon

Ergosterol is the principal sterol in fimgi and an important component of cell
membranes. This sterol has been previously used to measure fungal growth in foods. In
this project, ergosterol was derivatized with hemisuccinate (Erg-HS) and conjugated to
bovine serum albumin (Erg-HS-B SA). Six white female New Zealand rabbits were
immunized by subcutaneous injections of Erg-HS-BSA conjugate followed by four
booster injections. An indirect ELISA was used to assess titers. Although titers as high as
6400 were detectable, the antibody was not useful in detecting free ergosterol via
inhibition in a competitive indirect ELISA. The antibody was evaluated for applicability in
the ELICT for a variety of molds and yeasts but failed to detect presence of ergosterol in
the membranes of these organisms. Thus, although antibodies showed high specificity for

Erg-HS-BSA conjugate, they could not detect free ergosterol.

49

INTRODUCTION

Molds are distributed widely in soil, water, air, and food. They are responsible for
many cases of food spoilage in food products with reduced water. activity (aW 0.65-0.90)
or low pH at a wide range of temperatures. Fungal deterioration of cereals can take place
on the growing crop or in storage. The results can be a direct damage to grain because
production of dangerous mycotoxins.

Examination of food to determine its quality requires mycological techniques such
as direct and dilution plating. Plating techniques are based on enumeration of viable parts
of the organism. Those methods besides being time consuming and inaccurate, require
highly trained staff and only detect viable organisms. As an alternative, the Howard mold
count has been used by the food industry for detection of molds since 1916 (Howard,
1911). Microscopic examination of a representative sample is required, so consequently
this method requires special training. Also, this method lacks sensitivity, is imprecise and
time consuming.

Several methods to detect constituents of fungal cell walls have been suggested.
For example colorimetric methods for chitin determination have been developed for
different agricultural commodities (Donald et al., 1977; Whipps et al., 1980; Zak, 1976;
Becker et al., 1977). A high performance liquid chromatographic method was developed
by Lin and Cousin (1985) to detect glucosamine, a product that is released under acid,
alkaline or enzymatic hydrolysis of chitin, using derivatization by o-phthalaldehyde. A
new, faster, more inexpensive and sensitive method is needed. Thus, recently, enzyme-
linked immunosorbent assays (ELISAs) have been developed for mold detection using

species such as Penicillium, Mucor, and F usarium (Notennans et al., 1985, 1986; Lin et
50

51

al., 1986, 1987) but the specificity of these antibodies do not allow quantification of all
molds.

Rationale. Since ergosterol is the principal sterol in firngi and is present in
membranes, it is very suitable as a screening target. HPLC has been described to
determine ergosterol levels (Seitz et al., 1977) but this methodology needs clean-up and
extraction steps prior to analysis. Therefore, the development of a ELISA method to
determine ergosterol content to detect mold contamination in agricultural commodities
would be very useful.

The objective of this research was the production of antibodies for ergosterol and
their application in a competitive ELISA. Different carrier proteins have previously been
used before including keyhole limpet hemocyanin [KLH] and thyroglobulin [TG] by
Reynolds (1990), and cholera toxin [CT] by Dr. Azcona-Olivera [personal
communication, 1990] without positive results. The antigen used here consisted of
ergosterol conjugated to BSA (derivatizing first with hemisuccinate). Conjugation was
performed using the hydroxyl group on the terminal portion of the ergosterol outer ring
(the same group used when conjugated to KLH, TG and CT). However production of a
cyclic Diels-Alder adduct to protect double bounds in ergosterol structure, prior to
derivatization and conjugation, was the main difference between this new immunogen
(Erg-HS-B SA) and those used before (Erg-KLH, Erg-TG and Erg-CT). In this manner,
native ergosterol structure could not be modified afier conjugation. The results suggested
that although antibodies could be generated showing high specificity for Erg-HS-BSA

conjugate, they could not detect free ergosterol.

52

MATERIAL AND METHODS

Chemicals and reagents. All inorganic chemicals and organic solvents were
reagent grade or better and purchased from Aldrich Chemical Company (Milwaukee, WI)
or Sigma Chemical Company (St. Louis, MO), unless otherwise noted.

Preparation of immunogen. Ergosterol (Figure 18A) was derivatized
(hemisuccinate) (Figure 18C) and conjugated, using bovine serum albumin (BSA) (Figure
18D,E) as carrier protein, for use as an immunogen by Dr. M. Pallmer (Neogen Corp.,
Lansing, MI). Prior to derivatization and conjugation, ergosterol was treated with 4-
phenyl-1,2,4-triazoline-3,5-dione (PTAD) to protect 5,7-dien group, resulting in the
formation of a cyclic Diels-Alder adduct as reported by Anastasia et al. (1979) (Erg*)
(Figure 18B).

The presence of a hydroxyl group on the terminal portion of the ergosterol outer
ring provided favorable conditions and was therefore chosen for hemisuccinate
derivatization. Wei et al. (1971) described the basic procedure which was adopted for the
derivatization of Erg“ to Erg*-hemisuccinate (Erg*-HS). The formation of a
hemisuccinate chain attached to the former hydroxyl site was achieved through the
reaction of succinic anhydride with ergosterol. Pyridine acted as an electron acceptor in
the reaction. A carboxyl group on the terminus provided a reactive group for conjugation.
In a typical experiment, 252 mg (2.5 mM) succinic anhydride was briefly mixed until
dissolved with 800u1 pyridine in a small reaction vessel. Ten mg (2511M) Erg“ were added
and the mixture was refluxed for 60 min, then allowed to cool for approximately 5 min.
Pyridine was removed by submerging the reaction vessel in a boiling water bath and
applying a stream of nitrogen. Pyridine has a distinctive odor which aided in determining
when the compound had completely evaporated. Excess succinic anhydride was removed

by partitioning the mixture in equal volumes of water and chloroform phase, succinic

53

HO HO

A) Ergosterol

\
Pb

  
  

B) Erg‘. [Cyclic Diels Alder Adduct]
(Anastasia et al., 1979)

E) Erg-HS-BSA

Figure 18. - Structure of A) ergosterol, B) ergosterol" (cyclic Diels Alder adduct),
C) ergosterol*-hemisuccinate derivative, D) ergosterol"-hernisuocinate—BSA
conjugate. and E) ergosterol-hemisuccinate-BSA conjugate (immunogen).

54

anhydride partitioned into the water phase. Erg"-HS was dried under nitrogen and
dissolved in 0.5 ml benzenezacetone (70:30, v/v).

Low pressure chromatography on silica gel was used to separate Erg*-HS from
the unreacted Erg". A glass chromatographic column was packed with approximately 20 g
silica gel which was slurred in solvent (benzene:acetone, 70:30 v/v) and poured into the
column. A high pressure pump coupled to a pulse dampener was used to tightly pack silica
gel by passing solvent for several minutes through the column. Pressure was lowered fi'om
initially 90 p.s.i. to approximately 60 p.s.i. (7 ml/min) before loading the reacted mixture.
Because Erg*-HS is more polar than Erg“, free Erg" eluted first. Fractions were spotted
on silica gel TLC plates and developed in benzenezacetone (70:30). Compounds were
visualized by spraying with sulfirric acidzacetic acid (50:50). Fractions containing Erg*-HS
were pooled and dried under nitrogen. Erg*-HS concentration was determined by
absorbance in absolute ethanol at 282 nm . Typical recovery of Erg*-HS from the original
amount of pure Erg“ was approximately 70%.

An activated N-hydroxysuccinimide ester was used to conjugate Erg*-HS to
protein (Bauminger et al., 1973). Carboxyl-containing hapten, dicyclocarbodiimide (DCC)
and N-hydroxysuccinimide (molar ratio 1:1:2) were reacted in dimethyl formamide
(DMF). The removal of dicyclohexylurea, a byproduct of the reaction, was done by
centrifugation. N-Hydroxysuccinimide (NHS) esters bind only to amino groups, avoiding
cross linking of protein (via carboxyls) when carbodiimide alone is used. Two-fold excess
of NHS insured complete reaction of DCC to avoid cross linking. In a typical experiment,
2.86 mg DCC (13.9uM, 206 M.W.) and 3.19 mg NHS (27.8 [AM 115 M.W.) were mixed
together in 100 pl DMF and stirred at room temperature for several minutes. After drying
under nitrogen, 6.7 mg Erg*-HS (13.9uM, 497 M.W.) were mixed with 100 pl DMF.
Erg*-HS in DMF, and DCC and NHS in DMF were added together and mixed for an
additional 30 min at room temperature. This was allowed to settle (or centrifiiged briefly if

necessary) and supernatant was slowly added dropwise to each of two vials containing 2.5

55

mg protein (BSA) dissolved in 200 pl 0.1 N sodium bicarbonate. The solution was stirred
for 2 h at 4°C and then dialyzed against 50 mM sodium phosphate bufi‘er (pH 8.0) for 24
h. After conjugation to BSA, Erg*-HS-BSA (0.5g) in tetramethylguanidine (5 ml) was
heated at reflux for 15 min to give the structure of Erg-HS-BSA, which was used as the
immunogen (Fig. 18E).

Ergosterol-horseradish peroxidase (HRP) conjugation. Ergosterol was
derivatized to 6-amino hexanoic acid using the hydroxyl group on the terminal portion of
the ergosterol outer ring (Wei et al., 1971). Tert-butoxycarbonyloxyphthalimine (t-BOC)
was used as protective group, being later removed and replaced by HRP. In a typical
experiment, 3 mg of ergosterol derivative were dissolved in 75 pl of dimethylformarnide
(DMF) and 25 pl of distilled water. To cleave off the t-BOC protecting group, the pH was
brought down to 6 using 1M HCl (approximately 100 pl) and then pH was brought up to
pH 8 using 1M NaOH (approximately 100 pl). This solution was added to 30 mg of HRP
dissolved in 7 ml of distilled water containing 17 mg of l-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC) and 10 mg of N-hydrosuccinimide (NHS) and incubated for 1
h at room temperature with mixing. This new solution was kept overnight at 4°C avoiding
light. Then, it was dialyzed 3 times against saline 0.85% for at least 4 h each at 4°C. The
solution was aliquoted in 100pl vials and stored at 4°C in dark bottles.

Animal immunization. White female New Zealand rabbits were immunized by
subcutaneous injections (so) with Erg-HS-BSA conjugate at 4 weeks intervals. Three of
these rabbits were immunized using Freund's adjuvant and the other three using Hunter's
adjuvant (Titer max) (500 pg Erg-HS-BSA/dose). For those immunized with Freund's
adjuvant, the first injection consisted of 1 ml of conjugate in a 1:1 ratio of saline and
Freund's complete adjuvant, subsequent doses consisted of 1 ml of conjugate in saline and
Freund's incomplete adjuvant (1:1). For those immunized with Hunter‘s adjuvant, all the
doses consisted of 200 pl of conjugate in a 2:1 ratio of saline and Hunter‘s adjuvant. Time

intervals were 4 weeks between doses. Rabbits were bled from lateral marginal ear vein

56

approximately 2 weeks after each injection, and serum was obtained afier overnight
incubation of blood at 4°C and centrifugation at 1,000 x g for 15 min. Immunoglobulins
were purified by 33% saturation with ammonium sulfate (Hebert et al., 1973).

Enzyme linked immunosorbent assay (ELISA). For antiserum titration, wells of
polystyrene microtiter plates (Immunolon 4 Removawells, Dynatech laboratories,
Alexandria, VA.) were coated overnight ( 4°C) with 100 id or Erg-HS-BSA (10 pg/ml) in
0.1 M sodium carbonate-bicarbonate buffer (pH 9.6). Plates were washed four times with
300 pl of 0.02% (v/v) Tween 20 in PBS (PBS-Tween). Wells were blocked for 30 min at
37°C with 300 ul of 1% (w/v) BSA in PBS (BSA-PBS) and then washed four times with
PBS-Tween. Serially diluted serum (50pl) was then added to each well and incubated for
1 h at 37°C. Unbound antibody was removed by washing four times with PB S-Tween, and
100 pl of goat anti-rabbit IgG-HRP conjugate (diluted 1:1000 in BSA-PBS) was added to
each well. Plates were incubated for 30 min at 37°C, washed eight times with PBS-Tween,
and bound peroxidase detemrined with ABTS substrate as described previously by Pestka
et al., (1982). The absorbance at 405 nm was recorded by a Vrnax Kinetic Microplate
Reader with computer control (Molecular Devices' SOFT max) (Molecular Devices
Corporation, Menlo Park, CA). Titer of each serum was arbitrarily designated as the
maximum dilution that yielded at least twice the absorbance of the same dilution of
preimmune serum (NIS).

A competitive indirect ELISA (CI-ELISA) was used to assess the presence of
specific ergosterol antibodies in rabbit sera. Microtiter plates were coated and blocked as
described in the antiserum titration procedure. A 50 pl volume of ergosterol (stock
solution 1 mg/ml in ethanol or DMF), dissolved in PBS to give concentrations from 100 to
10,000 ng/ml, was then simultaneously incubated with a 50 pl volume of antiserum
(diluted 1:200 to 1:6400 fold in PBS, depending on the serum used) over the Erg-HS-
BSA solid phase for 1 h at 37°C. Bound antibody was determined by the addition of anti-
rabbit IgG-HRP conjugate as described above.

57

A competitive direct ELISA was developed and used to determine also the
presence and specificity of the ergosterol antibodies in the different rabbit sera. Plates
were coated overnight by air-drying at 40°C with 125 pl of sera produced in 0.1M sodium
carbonate-bicarbonate buffer (pH 9.6). Afier washing and blocking, 50 pl of fi'ee
ergosterol (stock solution 1 mg/ml in ethanol or DMF), dissolved in PBS to give
concentrations from 100 to 10,000 ng/ml, and 50 pl of Ergosterol-HRP were added
consecutively to each well. Afier 1 h of incubation at 37°C, plates were washed and bound
peroxidase was determined with 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)
(ABTS) substrate as described previously (Pestka et al., 1982). Absorbance was read at
405 nm.

Enzyme-linked immunocytochemical technique (ELICT). The method of
Lawellin et al. (1977) was used. Fixative solution was composed of: 20 g of para-
forrnaldehyde to which 150 ml of a double-filtered, saturated aqueous solution of picric
acid was added. Dissociation of the para-formaldehyde into formaldehyde in the strong
picric acid solution was obtained by heating (60°C) and on alkalinization of the mixture
with drops of 0.6N sodium hydroxide in water. A clear solution was obtained, which was
filtered, allowed to cool, and then made up to one liter with a 0.02M sodium phosphate
buffer (3.31 g NaH2P04-H20, 33.77 g Na2HP04-7H20, one liter distilled water, pH 7.3).
The fixative could be prepared in large quantities because it is very stable and withstands
exposure to light at room temperature for 12 months without deteriorating.

Mycelia from Aspergillus, Mucor, F. monilrforme M 5982, F. proliferatum M
5956 and yeast cells of Saccharomyces cerevisiae ATCC 49613 were obtained and fixed
in phosphate-buffered picric acid-formalin. After treating mycelia or cells (50 pl) with
fixative (100pl) for 8 h, samples were spun at 12,000 rpm in a clinical centrifuge and the
excess fixative removed. Vials were washed twice with 0.01M PBS for 30 min at room
temperature, centrifuged again and excess of PBS was removed. Ergosterol antibody fi'om

sera (diluted 1:50 with 1%[w/v]BSA-PBS)(100 pl) was added to the mycelia and cells. A

58

control was made without antibody. Samples were incubated for 24 h at room
temperature. Vials were centrifuged and supernatant was removed. Pellets were washed
with PBS-BSA twice by incubating 30 min at room temperature and centrifidgation. Afier
supernatant was removed, 100 pl of anti-rabbit IgG-HRP conjugate (diluted 1:50 in PBS-
BSA) was added to each vial and incubated for 3 h at room temperature. Samples were
washed with PBS-BSA and, after centrifuging, mycelia and cells were spread onto slides.
One drop of this diaminobenzidine solution (diaminobenzidine 0.005% in 0.05M TRIS
[3.02g TRIS BASE, 500 ml distilled water and pH 7.6], pH 7.6 with 0.001% H202) was
added and slides were covered with microscope cover glass. Preparations were sealed
with nail-polish and observed under a Nikon (Garden City, NY) brightfield and
fluorescence microscope equipped with Polaroid M-100 camera and Polaroid 667 film

(Polaroid corporation, Cambridge, MA).

59

RESULTS AND DISCUSSION

Rabbits were immunized by subcutaneous injections of an immunogen prepared by
conjugation of ergosterol to BSA. Four booster injections were performed at 4 weeks
intervals. Two different adjuvants were used (Freund's and Hunter's) as nonspecific
stirnulators of the immune response to induce a strong antibody response to soluble
antigens. These adjuvants have substances designed to form a deposit protecting the
immunogen from rapid catabolism, and other components that stimulate the immune
response nonspecifically. Rabbits were bled 2 weeks after each injection. Serum was
obtained and titers were assessed for each serum collected using an ELISA. Three
different blocking agents were used in this assay: 1 % polyvinyl alcohol-1 % gelatin
(PVA), Tween 20 and 1 % bovine serum albumin (BSA). The purpose to test these
blocking agents was the confusing results obtained for titration when ovalbumin was used
(one of the typical blocking agents generally used for ELISA). Moreover, the use of BSA
could reveal whether or not the antibody produced had any affinity for the protein used as
carrier in the immunogen conjugate. Best results were obtained using BSA. BSA and Erg-
HS-BSA were used as coating solutions (solid phase). Binding of antisera yielded higher
levels in Erg-HS-BSA coated wells than in BSA coated wells (Figures 21 and 22). The
maximal O.D. measured when testing preimmune sera (control) was 0.1-0.2, while for the
different antisera the OD. reached values ranging from 1.0 to 3.0 (Figures 19 to 21).
Although high titers were shown by different antisera (Figures 19, 20 and 21), inhibition of
binding by free ergosterol (competitive indirect ELISA)(Table 3) was not evident for any
of the antisera. Highest titers were always shown by rabbits immunized using Freund's
adjuvant (i.e. Figure 21, rabbit numbers 1F and 2F). Competitive direct ELISA results
were also always negative (Table 3). Inhibition of binding by free ergosterol was not
produced either by CD-ELISA or CI—ELISA, even though high titers were detected in

sera.

60

In conclusion, antibodies produced against Erg-HS-BSA were specific for Erg-
HS-BSA solid phase when compared to BSA (Figures 21 and 22). However, those
antibodies were not specific for free ergosterol. The immunogen prepared (Erg-HS-BSA)
may produce antibodies specific for the bridge between Erg-HS or carrier protein. This
would explain the observed binding to Erg-HS-BSA when used as solid phase with the
fiee ergosterol not being able to inhibit the binding. Also it is possible that Ergosterol was
decomposed during one or more of the derivatizing and conjugating steps, thus forming a
new compound. The antisera produced might be more specific for such compounds than
for ergosterol.

To further ascertain whether antibodies produced against Erg-HS-BSA
immunogen had specificity for ergosterol, ELICT was performed for five different
microorganisms: S. cerevisiae ATCC 49613, Mucor, Aspergillus, F. monilrfonne M 5982,
and F. proliferatum M 5956. However, visualization of ergosterol in the membranes was
not successful, thus indicating possibly absence of affinity between Erg-HS-BSA antibody
produced in sera and ergosterol present in microorganism membranes. It is possible that
fungal cell wall prevented penetration of the antibody fi'om reaching the membrane, the
place where ergosterol is found. The cell wall provides the interface between the organism
and its environment. It confers shape to the cell and this is a function of its rigidity which
in turns affects the osmotic integrity of the cell. One of its functions would be to indirectly
affect the intracellular concentration of ions and solutes by restricting the water content of
the cell, thus, aqueous solution ( as fixative, antibody solution, etc.) would not be allowed

to penetrate through the wall to reach the membrane.

61

CONCLUSIONS

Although titers as high as 6400 againts Erg-HS-BSA were detectable, the antibody
produced was not useful in detecting free ergosterol via inhibition in a competitive indirect
and/or competitive direct ELISA. Similarly attempts to demonstrate its applicability in the
ELICT were not successful. Thus, antibodies could be generated that showed high

specificity for Erg-HS-BSA conjugate but not free ergosterol.

62

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LIST OF REFERENCES

LIST OF REFERENCES

Abouzied, M.M. and J .J . Pestka (1994). Simultaneous screening of firmonisin Bl,
aflatoxin B1 and zearalenone by Line Immunoblot: a computer-assisted multianalyte assay
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