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This is to certify that the

dissertation entitled

Recombinant Antibodies and Peptide
Mimotopes to FusariumiMycotoxins

presented by
Qiaoping Yuan

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Botany and Plant Pathology
- Environmental Toxicology

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ma clumps-m4

 

RECOMBINANT ANTIBODIES AND PEPTIDE MIMOTOPES
TO FUSARIUM MYCOTOXINS

By

Qiaoping Yuan

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology,
and Institute of Environmental Toxicology

1998

ABSTRACT

RECOMBINANT ANTIBODIES AND PEPTIDE MIMOTOPES
TO FUSARIUM MYCOTOXINS

By
Qiaoping Yuan

Zearalenone, fumonisins and trichothecene mycotoxins are a group of
secondary metabolites produced by toxigenic strains of Fusarium fungi. These
mycotoxins are toxic to both human and animals; Some are also phytotoxic.
Modern immunology and molecular biology provide novel approaches to develop
low-cost antibodies and alternative antigens with desirable affinity and specificity
for mycotoxin detection, neutralization, and detoxification as well as the
improvement in plant resistance to mycotoxin associated plant diseases. In this
thesis, phage display technology was explored to develop recombinant antibodies
and peptide mimotopes for Fusarium mycotoxins. Firstly, the heavy and kappa light-
chain variable region genes of an anti-zearalenone hybridoma cell line were
isolated by polymerase chain reaction and joined by a DNA linker encoding peptide
(Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was
cloned and expressed as a fusion protein with an antibody recognition sequence (E-
tag) and phage M13 coat protein 93p in Escherichia coli TG1. To facilitate the
cloning of genes for variable heavy chain, variable kappa light chain, and the
assembly of scFv DNA fragment, a new phage display vector, pEY.5, was also
constructed. Through affinity panning-elution, high affinity scFv phage antibodies
were enriched and selected. Nucleotide sequence analyses revealed that the VH

portion of the cloned scFv antibody contributed more to the specificity than the VK

portion. The potential of using the soluble scFv antibody for routine screening of
zearalenone and its analogues was demonstrated. With a similar approach,
attempts were made to generate scFv antibodies from mice immunized with
fumonisin B,-cholera toxin conjugate immunized mice. However, no specific scFv
antibody clone was selected. An alternative approach was suggested for the
cloning of functional FB1 specific scFv antibodies. Secondly, the anti-zearalenone
scFv DNA fragment was cloned into a plant transformation vector and transferred
into Ambidopsis immature seeds by vacuum infiltration via Agrobacterium
tumefaciens mediated plant transformation. Functional anti-zearalenone scFv
plantibody was expressed. The plantibody exhibited a high zearalenone-binding
affinity comparable to bacteria-produced scFv antibody and the parent monoclonal
antibody. Finally, from a phage displayed random 7-mer peptide library, two highly
homologous heptapeptide sequences, SWGPFPF and SWGPLPF, were identified
that specifically bound a deoxynivalenol-specific monoclonal antibody. In contrast
to deoxynivalenol’s toxic effects, the peptide did not elicit apoptosis in bone marrow
cells, neither did it inhibit protein synthesis in a cell-free translation system. Rather,
the peptide mimotope showed an antagonism to deoxynivalenol-induced protein
synthesis inhibition. These peptide mimotopes were also evaluated as

immunochemical reagents for deoxynivalenol immunoassay.

To my dear parents, my wife, and my lovely twin daughters

iv

ACKNOWLEDGMENTS

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

I wish to thank Dr. Hui-Ren Zhou, Dr. James R. Clarke, Ms. Rebecca
Uzarski, Mr. Wenqi Hu, and many others from Dr. Hart’s and Dr. Pestka’s
laboratories for their invaluable suggestions, practical help and friendship.
Acknowledgment also goes to Dr. Shenyang He and his students for their help with
plant transformation and to Mr. Frank E. Klein at the Neogen Company for his help
in making deoxynivalenol conjugates.

Most importantly, I would like to thank my wife Youlan Chen, and my
daughters, Jing Yuan and Lin Yuan, for their love, support, patience, and
understanding during all of these years. I am also very grateful to my father, Junxi
Yuan, my mother, Meichun Xiang, my brother Xuewen Yuan and my sister Hongxia

Yuan, for their continuous love and support in my life.

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................... x
LIST OF FIGURES .................................................................................................... xi
LIST OF SYMBOLS AND ABBREVIATIONS .......................................................... xvii
INTRODUCTION .................................................................................................................... 1
PART I. LITERATURE REVIEW ............................................................................. 4
MYCOTOXINS ...................................................................................................... 5
Zearalenone .................................................................................................... 6
Fumonisins .................................................................................................... 12
Tn'chothecenes .............................................................................................. 16
Analytical methods for mycotoxin detection .................................................. 19
RECOMBINANT ANTIBODIES AND PEPTIDE MIMOTOPES ........................... 22
Native antibody .............................................................................................. 22
Recombinant antibody and phage display ..................................................... 23
Peptide mimotopes ........................................................................................ 29
PLANTIBODY ..................................................................................................... 31

PARTII. MOLECULAR CLONING. EXPRESSION AND
CHARACTERIZATION OF A FUNCTIONAL SINGLE-CHAIN Fv

(scFv) ANTIBODY TO THE MYCOTOXINZEARALENONE ................... 36
ABSTRACT ......................................................................................................... 37
INTRODUCTION ................................................................................................. 39
MATERIALS AND METHODS ............................................................................ 41

Materials, strains and general methods ......................................................... 41
mRNA isolation .............................................................................................. 43
cDNA synthesis, PCR amplification and scFv cloning ................................... 43
ScFv antibody expression and panning-elution selection .............................. 46
Competitive indirect ELISA (Cl-ELISA) ........................................................ 48
SDS-PAGE and Western blot analysis .......................................................... 50
Sequence analysis ........................................................................................ 51
CI-ELISA for spiked corn extracts ................................................................ 51
Nucleotide sequence accession numbers ..................................................... 52

vi

PAI

 

RESULTS ......................................................................................................... 53

PCR amplification .......................................................................................... 53
Phage display of scFv antibody and panning-elution selection ..................... 55
Soluble scFv antibody production and characterization ................................ 56
SDS-PAGE and Western blot analyses ........................................................ 60
Sequence analysis ........................................................................................ 60
Application of scFv OY1 .5toa spiked corn extract ....................................... 64
DISCUSSION ..................................................................................................... 67

PART III. ATTEMPTS TO CLONE SINGLE-CHAIN FV (SCFV) ANTIBODIES

AGAINST FUMONISIN B1 FROM IMMUNIZED MICE ............................. 71
ABSTRACT ......................................................................................................... 72
INTRODUCTION ................................................................................................. 73
MATERIALS AND METHODS ............................................................................ 76

General procedures ....................................................................................... 76
Preparation of immunogens .......................................................................... 76
Animal immunization ..................................................................................... 77
ELISA ............................................................................................................ 79
Spleen mRNA isolation and cDNA synthesis ................................................ 80
PCR amplification and scFv cloning .............................................................. 80
Affinity selection of phage-displayed anti-PB, scFv antibodies ...................... 81
RESULTS AND DISCUSSION ............................................................................ 84
Mouse immunization and polyclonal antibodies ............................................ 84
Construction of phage-displayed scFinbrary ................................................ 86
Affinity selection of phage-displayed scFv antibodies ................................... 88
CONCLUSION .................................................................................................... 92

PART IV. EXPRESSION OF A FUNCTIONAL ANTI-ZEARALENONE
SINGLE-CHAIN FV ANTIBODY IN TRANSGENIC ARABIDOPSIS

PLANTS .................................................................................................. 93
ABSTRACT ......................................................................................................... 94
INTRODUCTION ................................................................................................. 95
METHODS AND MATERIALS ............................................................................ 97

General .......................................................................................................... 97
Constmction of scFv cloning vector ............................................................... 97

vii

Anti-zearalenone scFv antibody cloning in pEY.5 ........................................ 99

Construction of plant scFv expression plasmids ........................................... 99
Plant transformation ................................................................................ 100
Expression of scFv plantibody and ELISA analysis ..................................... 101
Protein analysis ........................................................................................... 102
ScFv antibody cytolocation with immunogold labeling ................................. 103
RESULTS .......................................................................................................... 105
Cloning of anti-zearalenone scFv in pEY.5 ................................................. 105
Expression of anti-zearalenone scFv plantibody ......................................... 107
The effects of methanol and low pH on plantibody ..................................... 1 1 1
Localization of anti-zearalenone scFv plantibody ........................................ 113
DISCUSSION .................................................................................................... 115

PART V. IDENTIFICATION OF MIMOTOPE PEPTIDES WHICH BIND TO
THE MYCOTOXIN DEOXYNIVALENOL-SPECIFIC

MONOCLONAL ANTIBODY ............................................................... 1 19
ABSTRACT ....................................................................................................... 120
INTRODUCTION ............................................................................................... 122
MATERIALS AND METHODS .......................................................................... 126

Reagents ..................................................................................................... 126
Phage-display peptide library and amplification .......................................... 128
Phage selection by panning-elution selection ............................................. 128
Titration and amplification of panning-elution selected phage ..................... 131
Competitive ELISA with phage-displayed peptide ...................................... 131
DNA sequencing ......................................................................................... 132
Translational fusion with bacterial alkaline phosphatase (AP) .................... 133
CD-ELISA with DONPEP—AP fusion protein ................................................ 134
CD-ELISA using peptide C430 and its HRP conjugate ............................... 134
Immunization of animals with C430-BSA conjugate .................................... 135
Cytotoxicity and effects on protein synthesis in vitm ................................... 135
RESULTS .......................................................................................................... 138
Panning-elution selection of specific phage ............................................... 138
Application of DON mimotope peptide in competitive ELISA ...................... 140
CD—ELISA for DON spiked wheat extract ................................................... 145
Immunization with C430-BSA conjugate ..................................................... 147
Cytotoxicity and effects on protein synthesis in vitro .................................. 147
DISCUSSION .................................................................................................... 152

viii

SUMMARY ............................................................................................................. 1 58

APPENDICES: BUFFERS AND MEDIA ................................................................ 159
APPENDIX A: COMMONLY USED BUFFERS ............................................. 160
APPENDIX B: COMMONLY USED MEDIA ................................................. 163

LIST OF REFERENCES ....................................................................................... 164

ix

Table 1.1.
Table 1.2.

Table 2.1.

Table 2.2.

Table 4.1 .

Table 5.1.

LIST OF TABLES

The genes and proteins of fd bacteriophage ...................................... 25
Antibody-derived molecules produced in transgenic plants ................ 35
Sequences of oligonucleotide PCR primers for PCR

amplification of V", VK, and linker DNA ............................................. 45
Comparison of cross-reactivities between scFv antibody and

mAb toward zearalenone analogues ................................................. 59
Effect of pH environment on antibody binding activity ...................... 1 12

50% binding inhibition of DON-HRP and C430-HRP by DON
and C430 in competitive ELISA with monoclonal antibody 6F5
coated in microtiter wells ............................................................... 143

LIST OF FIGURES

Figure 1.1. Structures of zearalenone and analogues showing structural
similarities with estradiol .................................................................... 7

Figure 1.2. Structures of known fumonisins and Altemaria altemata f. sp.
lycopetsici (AAL) toxin, showing stmctural similarities with
sphingosine (Norred, 1993) ............................................................ 14

Figure 1.3. Structure and numbering system of some naturally identified
trichothecene mycotoxins (modified from Ueno, 1983) .................. 17

Figure 1.4. Antibody model structures showing subunit composition and
domain distribution along the polypeptide chains. Fragments
generated by proteolytic cleavage and [or recombinant
technology appear in the shaded area (Phannacia Biotech) .......... 24

Figure 2.1. Preparation of zearalenone immunogens through oxime
chemistry (based on Thouvenot and Morfin, 1983) ......................... 42

Figure 2.2. Panning-elution selection procedure for anti-zearalenone scFv
phage in zearalenone-ovalbumin (ZEN-OVA) coated Immunlon-
4 microtitetwells .............................................................................. 47

Figure 2.3. VH and VK amplification and assembly of scFv. Lanes are: (1):
VH marker (pUC18NH, EcoR I/Xba I digested, Pharrnarcia
Biotech); (2): VH PCR product; (3): VK PCR product; (4): Linker
PCR product; (5): Linker-VK PCR product; (6): VH-Linker-VK
(scFv) PCR product; (7): scFv marker (pUC18/A10B, Sfi l/ Not
I digested, Pharrnacia Biotech) ...................................................... 54

Figure 2.4. pQY1 construct. PCR amplified scFv was cloned into Sfil and
Not I sites of pCANTAB5E, a pUC119 based vector from
Phannacia Biotech. The linker DNA sequence was
5'GGTGGAGGCGG‘I'I'CAGGCGGAGGTGGCTCTGGCGGTG
GCGGATCG-3', which encoded a short peptide
[(Gly,Ser)3] ....................................................................................... 55

Figure 2.5. Zearalenone-binding activities of recombinant phage following
each round of panning-elution selection measured by Cl-ELISA
in zearalenone-ovalbumin coated Immulon-4 microtiter wells.
Bound phages were detected by horseradish peroxidase
conjugated sheep anti-M13 antibody .............................................. 57

xi

Figure

Figure

Figure

Fbum

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Figure

Figure 2.6. Sensitivity of soluble scFv antibodies tested in CI-ELISA in
zearalenone-ovalbumin coated Immulon-4 microtiter wells.
QY1.1 and QY1.2 ( no dilution) were from preselection; QY1.5
(5x dilution) was from first round of panning-elution selection;
QY1.12 (10 x dilution) was from second round of panning-
elution selection; mAb (supernatant of hybridoma cell culture)
was diluted 5 times. Bound soluble scFv antibodies were
detected by mouse anti-E tag antibody, followed by horseradish
peroxidase conjugated goat anti-mouse IgG. A: Absorbance
reading at 405 nm. B: Percent of binding inhibition determined
by dividing sample absorbance by control absorbance
x 100 ............................................................................................. 58

Figure 2.7. SDS-PAGE gel and Western blot analysis of scFv expression.
(A): Supematants of QY1 .1 (lane 1; 0.3 ml), QY1.2 (lane 2; 0.3
ml), QY1.12 (Iane3; 0.3 ml) and QY1.5 (lane 4; 0.6 ml)
concentrated in 10% trichloroacetic acid were subjected to SDS-
PAGE and stained with Coomassie brilliant blue G-250. (B):
Western blot ofduplicate SDS/PAGE gel ......................................... 61

Figure 2.8. Deduced amino acid sequences of VH and VK in scFv QY1.5.
Complementary-determining regions (CDR) and framework
regions (FW) are indicated ................................................................ 63

Figure 2.9. Zearalenone sensitivities in spiked corn extracts with monclonal
antibody and soluble scFv antibody QY1.5 measured by CI-
ELISA in zearalenone-keyhole limpet hemocyanin conjugate
coated lmmulon-4 microtiter wells. Both scFv QY1.5 and mAb
were diluted 1:4 in PBS containing 10% nonfat dry milk.
Zearalenone in ground corn was extracted with 5 volumes
(wt/vol) of methanol-water (70:30) and the extracts were then
diluted 1:1 with PBS .......................................................................... 65

Figure 2.10. Effects of methanol on the binding of mAb and scFv QY1.5 to
zearalenone-keyhole limpet hemocyanin conjugate. lmmulon-4
microtiter wells were coated with zearalenone-keyhole limpet
hemocyanin conjugate. Bound soluble scFv antibody was
detected by mouse anti-E tag antibody, followed by horseradish
peroxidase conjugated goat anti-mouse IgG. Bound monoclonal
antibody was detected by horseradish peroxidase conjugated
goat anti-mouse IgG .......................................................................... 66

Figure 3.1. Preparation of fumonisin B1 conjugates with glutaraldehyde
linkage via a free amino group (Pestka et al., 1995) ........................ 78

xii

 

 

 

 

 

 

 

Fig

Fig

Fig

Fic

Fig

Figure 3.2. Polyclonal anti-FB, antibody production from mice immunized
with FB1-cholera toxin (FB1-CT) and FB1-keyhole limpet
hemocyanin (FB,-KLH) conjugates (Each group had one
representative out of eight mice). Sera were obtained one week
after the second boost with the immunogens. A: Titration of
antisera measured in FB,-ovalbumin (FB,-OVA) conjugate
coated Immulon-4 microtiter wells. Bound mouse antibodies
were detected by horseradish peroxidase conjugated goat anti-
mouse lgG. B: Competitive indirect ELISA for FB1 with the
antisera (diluted 1:60.000 and 1:3,200 respectively for FB,-KLH
and FB,-CT immunized mice) ........................................................... 85

Figure 3.3. PCR amplification of VH and VK genes from mouse splenic
mRNA and the assembly of scFv. Lanes are: (1): Linker PCR
product; (2): VK PCR product; (3): VH PCR product; (4): Linker-

VK PCR product; (5):VH-Linker-VK (scFv) PCR product; Lane M:
100-bp ladder DNA marker (Gibco BRL) ............................................ 87

Figure 3.4. Phage titers after panning-elution selection of the phage library.
Standard procedure was: coating: 1 pg/well of FB,-OVA
conjugates or OVA alone (in control wells); phage incubation: 60
min; bound phage elution: by PBS or 100 jig/"II of FB1 in PBS.
Modifications were different conjugates for coating (A), phage
incubation time (B), and FB1 concentration for elution (C) .............. 89

Figure 4.1. Map of phage-display vector pEY.5. (A). Restriction map with
unique restriction sites marked. (B). The nucleotide sequence of
linker (bold sequence) and the VH and VK cloning
sites (underlined)in pEY.5 ............................................................... 98

Figure 4.2. Sensitivity and specificity of anti-zearalenone scFv antibodies
measured by competitive indirect ELISA using zearalenone-
ovalbumin coated wells. Bound soluble scFv antibody was
detected by mouse anti-E tag antibody, followed by horseradish
peroxidase conjugated goat anti-mouse IgG. EY.5HL3 was
produced in E. coli and plantibody was
produced in Arabidopsis ................................................................ 106

Figure 4.3. The transgene in plant transformation constructs pQY4.1-1 (A)
and pQY4.4-1 (B). 3582: Can 35S promoter with a duplicated
enhancer; sp: plant pathogenicity—related protein PR-1b signal
peptide sequence; T,m and Tm: transcription lamination
sequences for tch and nos respectively; Pm: nos promoter,
Kn': kanamycin resistance gene .................................................... 108

xiii

I Figure 4

Figure .

Figure

Figure

 

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Figure

Figure

Figure 4.4. The germination of Arabidopsis tansformant seeds on
kanamycin selection medium plates. A. Seeds from un-
transformed control plants; B. Seeds from plants transformed
with pQY4.4-1, targeting scFv antibody to the apoplastic space
with signal peptide sequence. C. Seeds from plants transformed
with pQY4.1-1 , targeting scFv antibody to the cytoplasm ............... 109

Figure 4.5. SDS-polyacrylamide gel electrophoresis and Western blot
analysis of anti-zearalenone scFv plantibody expression.
Soluble proteins of Arabidopsis from un-transformed plants (lane
1), vector pKYLX71::35S2 transformed plants (lane 2), and
pQY4.4-1 transformed plants (lane 3) were subjected to SDS-
PAGE and stained with Coomassie brilliant blue G-250 (A).
Western blot of duplicate SDS-polyacrylamide gel detected by
anti-E tag antibody (B). Lane M: protein molecular size marker
with 10KDa-ladder ........................................................................ 110

Figure 4.6. Effect of methanol on the binding of anti-zearalenone
monoclonal antibody (mAb) and bacterial scFv antibody
(EY.5HL3) and plant scFv antibody (plantibody) to immobilized
zearalenone-KLH conjugate ........................................................... 112

Figure 4.7. Cytolocation of anti-zearalenone scFv plantibody by
immunogold labeling and transmission electron microscopy.
Leaf sections from plants expressing anti-zearalenone scFv
plantibodies (A, B) and non-transformed control plants (D) were
detected by mouse anti-E tag antibody followed by goat anti-
mouse lgG gold conjugate. (C): Leaf sections from non-
transforrned control plants were pre-incubated with anti-
zearalenone scFv antibody, then with mouse anti-E tag antibody
followed by goat anti-mouse IgG gold conjugate. CW, cell wall;
CY, cytoplasm, ICS, intercellular space; Arrows: gold labeling;
Magnification x 39,000 ............................................................... 114

Figure 5.1 . Preparation of deoxynivalenol (DON) conjugates via 3-hydroxyl
group (Pestka et al., 1995) ............................................................ 127

Figure 5.2. Panning-elution selection procedure for deoxynivalenol (DON)
mimotopes in anti-DON monoclonal antibody coated Immulon-4
microtiter wells ............................................................................... 130

Figure 5.3. Enrichment of mAb 6F5 specific phage. Phage were titered
after elution with DON (100 pglml) from mAb 6F5 coated
lmmulon-4 microtiter wells or from mouse IgG coated Immulon—4
microtiter wells in each round of panning-elution
selection ............................................................ 139

xiv

 

 

Figure

Figure

FIgUI'I

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Flgu

Flgu

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FIQL

Figure 5.4. Competition between phage-displayed mimotope peptides and
DON for binding to immobilized mAb 6F5 in CD-ELISA. Various
concentrations of free DON competed with an equal volume of
phage-displayed peptides (at constant concentration) for binding
to immobilized mAb 6F5. Bound phage peptide was detected by
horseradish peroxidase conjugated sheep anti-M13 IgG and
then measured by absorbance. DON-HRP was included as a
positive control ................................................................................ 141

Figure 5.5. Competition between synthetic peptide C430 and DON for
binding to mAb 6F5. (A). DON and synthetic peptide C430
compete with DON-HRP for binding to immobilized mAb 6F5.

(B). DON and synthetic peptide C430 compete with C430-HRP
for binding to immobilized anti-DON mAb 6F5 ................................ 142

Figure 5.6. Competition between individual amino acids in synthetic peptide
C430 and DON measured with CD-ELISA. Amino acids (at
various concentrations) competed with DON-HRP for binding to
immobilized mAb 6F5 ...................................................................... 143

Figure 5.7. CD-ELISA with DONPEP-AP fusion protein. Binding of
DONPEP—AP fusion protein to immobilized mAb 6F5 was
inhibited by free DON. DON-HRP was included as a positive
control ............................................................................................. 144

Figure 5.8. Application of C430-HRP and DONPEP-AP in DON
immunoassay (CD-ELISA) in a DON-spiked wheat extract.
Immulon-4 microtiter wells were coated with mAb 6F5 antibody.
DON-HRP was included as a positive control ................................ 146

Figure 5.9. The specificity of antibody from C430-BSA immunized mice.
DON and C430 at various concentrations were used to inhibit
the binding of antisera to immobilized DONPEP.2 phage. Bound
mouse antibodies were detected by horseradish peroxidase
conjugated goat anti-mouse lgG and measure by
absorbance .................................................................................... 148

Figure 5.10. Cytotoxicity to bone marrow cells after 18 hr incubation with
synthetic peptide C430 co-incubated with (white bar) or without
(hatched bar) DON (3.4 uM). Cell viability was determined using
a MTI' conversion assay .............................................................. 149

Figure 5.1 1 . Effects of DON (3.4 pM) and synthetic peptide C430 (3.4 uM) on
protein synthesis in vitro with rabbit reticulocyte Iysate. The

translation template was y-globulin mRNA (2 pg). “+” or “-”:
with or without added, respectively .............................................. 151

XV

 

 

 

 

 

 

Figure 5.

Figure 5.12. Possible mechanisms for the binding of DON mimotope
peptides to mAb 6F5 and for the immunogenicity of C430-BSA.
In Model 1, DON and DONPEP might bind to different portions
at the antigen binding pocket of mAb 6F5. After immunization
with DONPEP, only anti-DONPEP antibody is produced. In
Model 2, DON and DONPEP bind to mAb 6F5 in the same
fashion. But after immunization with DONPEP, only the epitope
which is different from DON elicits antibody response .................... 154

 

 

ABTS
AP“. 3‘
BCle
BSA:
CD-E
CDR:
Cl-El
CT: c
DON
EDTI
ELEl
ELIS
FB1 :

GC:
HPLI
HRP
ICSQ:
IPTC
KLH.

MBS
MS:
NBT
OVA
PAG
PCR
PBS
PEP
DNP
PMS

SCH

LIST OF SYMBOLS AND ABBREVIATIONS

ABTS: 2,2-azinobis(3—3-ethylbenzthiazoline sulfonic acid).
AP: alkaline phosphatase

BCIP: 5-bromo-4-chloro-3-indolyphosphate-p-toluidine.
BSA: bovine serum albumin.

CD-ELISA: competitive direct enzyme-linked immunosorbent assay.
CDR: complemetarity-determining region.

CI-ELISA: competitive indirect enzyme-linked immunosorbent assay.
CT: cholera toxin.

DON: deoxynivalenol.

EDTA: ethylenediaminetetraacetate.

ELEM: equine leukoencephalomalacia.

ELISA: enzyme-linked immunosorbent assay.

FB1 :fumonisin B1.

FW: framework region.

60: gas chromatography.

HPLC: high performance liquid chromatography.

HRP : horseradish peroxidase.

I050: the concentrations required for inhibition of binding by 50% ,
IPTG: isopropyI-[i-D-thiogalactopyranoside.

KLH: keyhole limpet hemocyanin.

mAb: monoclonal antibody.

MBS: m-maleimidobenzoyI-N-hydroxysuccinimide ester.
MS: mass spectroscopy.

NBT: nitroblue tetrazolium.

OVA: ovalbumin. '

PAGE: polyacrylamide gel electrophoresis.

PCR: polymerase chain reaction.

PBS: phosphate-buffered saline.

PEP: porcine pulmonary edema.

pNPP: p-nitrophenyl phosphate.

PMSF: phenylmethylsulfonyl fluride.

RACE: rapid amplification of cDNA ends.

scFv: single-chain Fv fragment.

SDS: sodium dodecyl sulfate.

TBS: Tris-buffered saline.

TCA: trichloroacetic acid.

TLC: thin layer chromatography.

TMB: 3,3',5,5'-tetramethyl-benzidine.

VH: variable region of heavy chain.

Vk: variable region of kappa light chain.

VL: variable region of light chain.

ZEN: zearalenone.

xvii

INTRODUCTION

Mycotoxins are a group of secondary metabolites produced by toxigenic
strains of fungi. Fusarium is one of the major fungal genera that contains species
capable of mycotoxin production. Zearalenone, fumonisins and trichothecenes are
Fusarium mycotoxins that are toxic to both human and animals and are commonly
found in grain and grain products infected by Fusarium spp. The presence of these
toxins in agricultural commodities is primarily governed by environmental and
biological factors. A major means of eliminating mycotoxins from human and animal
food is to detect and divert contaminated raw materials from feed and finished food
use.

Mycotoxin detection can be carried out using either conventional chemical
methods such as thin layer chromatography (TLC), gas chromatography (60), high
performance liquid chromatography (HPLC), and mass spectroscopy (MS) or
immunoassay techniques such as enzyme-linked immunosorbent assay (ELISA).
Immunoassay is commonly preferred to conventional chemical methods because
of its high specificity, sensitivity, simplicity, rapidity, and applicability both in the field
and the laboratory.

Consequently, immunoassays for zearalenone, fumonisins, and the
trichothecene deoxynivalenol (DON) have been developed in our laboratory with
both polyclonal and monoclonal antibodies. However, the limits of detection with the
existing antibodies to fumonisin and deoxynivalenol are not sufficiently adequate for

screening these compounds at <1 ppm in conventional immunoassays, while

detection at $0.1 ppm would be desirable in order for the agrifood industry to
anticipate problems in a growing season. Therefore, production of higher affinity and
low-cost specific antibodies against these Fusarium mycotoxins is desirable.
Immunoassay for deoxynivalenol encounters further problems because chemical
conjugation of DON to a carrier protein or an enzyme has low efficiency and the
conjugates are weakly immunogenic. Therefore, alternative reagents are needed
to replace deoxynivalenol as immunogens and immunochemical reagents.

With newly developed phage display technology, a specific clone for
antibody, or protein, or peptide can be selected from large repertoires because the
DNA sequence is linked with its expression product in a single phage particle. This
provides a powerful tool to clone mycotoxin antibody genes and to search
proteinaceous mimics for mycotoxins. Once the specific antibodies to mycotoxins
are cloned, the genes can also be introduced into plants. These plant-produced
antibodies (plantibodies) might be very useful to neutralize mycotoxins through
passive immunization of animals by feeding and to improve plant resistance against
mycotoxin-associated plant diseases.

The research in this dissertation was undertaken to address the above
rationales and is presented in five parts. Part I (Literature Review) reviews Fusarium
mycotoxins especially zearalenone, fumonisins and trichothecenes including their
chemical structures, toxicity, natural occurrence, and detection. The development
of recombinant antibodies, peptide mimotopes and plantibodies is also reviewed.
Part II (Molecular Cloning, Expression and Characterization of A Functional Single-

chain Fv Antibody to the Mycotoxin Zearalenone) describes in detail the molecular

 

 

 

 

during 1

(scFv) a

 

dmes
zearaler
(Attemp‘.
lmmunig
from m
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Transge '
anlizea

of a neii

Mimoioi

 

Monoclc
mimotoi
The am

SOme C

 

cloning (phage display and expression in bacteria) of a functional single-chain Fv
(scFv) antibody to the mycotoxin zearalenone from a hybridoma cell line. The use
of the scFv antibody for ELISA development and the application of ELISA for
zearalenone detection in spiked corn samples are also demonstrated. Part III
(Attempts to Clone Single-Chain Fv Antibodies Against Fumonisin B1 from
lmmunized Mice) explains efforts to produce scFv antibodies with phage display
from mice immunized with fumonisin B1-cholera toxin conjugate. Part IV
(Expression of A Functional Anti-Zearalenone Single-Chain Fv Antibody in
Transgenic Arabidopsis Plants) describes in detail the expression of a functional
anti-zearalenone scFv antibody in transgenic Arabidopsis plants. The construction
of a new phage-display cloning vector is also described. Part V (Identification of
Mimotope Peptides Which Bind to the Mycotoxin DeoxynivalenoI-Specific
Monoclonal Antibody) describes the identification of two deoxynivalenol
mimotope peptide sequences from a phage-display random 7-mer peptide library.
The application of these peptide mimotopes in deoxynivalenol immunoassay and

some of their bio-activities are also described.

PART I.
LITERATURE REVIEW

 

 

 

 

N,
apart oi
Casale,

dunng g

 

 

 

dunng l
includin
biologi
at haw
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In som
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effects:
and car
I
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VITUIen
1989;

SChO/br

 

MYCOTOXINS

Mycotoxins are a chemically diverse group of toxic compounds produced as
a part of secondary metabolism in a wide variety of filamentous fungi ( Pestka and
Casale, 1990). They often contaminate agricultural commodities priorto harvest and
during storage. These natural toxicants are elaborated in plant tissue infected
during the growing season or later in storage environments by common fungi
including species of Aspergillus, Penicilium and Fusarium. Environmental and
biological factors, particularly the regional weather during the growing season and
at harvest, determine the degree to which a mycotoxin will contaminate a
commodity (CAST, 1989). Because mycotoxins are resistant to food processing and
do not degrade at high temperatures, they enter the human and animal food supply.
In some cases, mycotoxins are passed on through animal feed to meat and poultry
foods (Pestka et al., 1995). Human and animal mycotoxicoses include diverse
effects such as gastroenteritis, hypetestrogenism, kidney dysfunction, neurotoxicity
and cancer (Pestka and Casale, 1990).

Although mycotoxins are defined as fungal secondary metabolites toxic to
animals, many of them are also phytotoxic and serve as virulence factors for plant
pathogenic fungi (Desjardins and Hohn, 1997). For example, there is a growing
body of evidence indicating that trichothecenes, including DON, can enhance the
virulence of plant-pathogenic species of Fusarium on plant hosts (Adams and Hart,
1989; Desjardins et al., 1989; 1992; Bruins et al., 1993; Wang and Miller, 1988;

Scholbrock et al., 1992; Atanassov et al., 1994; Proctor et al., 1995). Fumonisins

 

 

 

have a
concen
Fusani
zeanue

Mach,

uthofl

mycou

Zearai

undec
lNodur
and C
Woddi
Warns

zeara

have also been shown to cause necrosis and other disease symptoms at low
concentrations and are associated with high level of virulence in pathogenic
Fusarium (Lamprecht et al., 1994; Desjardins et al., 1995). The mycotoxin
zearalenone has also been reported to cause certain phytotoxicities (Vianello and
Maori, 1981; Wakulinski, 1989; Nedelnik, 1993).

Among the most important mycotoxins are zearalenone, fumonisins,
trichothecenes, aflatoxins, and ochratoxins (Pohland, 1993). The first three

mycotoxins are produced by Fusarium and will be reviewed in this chapter.

Zearalenone

Production and occurrence. Zearalenone [6-(10-hydroxy-6-oxo-trans-1-
undecenyIFB-resorcylic acid-lactone] (Figure 1.1) is an estrogenic mycotoxin
produced by the genus Fusarium after infection of corn and small grains (Pestka
and Casale, 1990). Species of Fusarium are extremely common in nature and
worldwide in distribution. They are active pathogens infecting a wide variety of host
plants. Since the first finding of zearalenone in Fusarium-infected corn in 1960s,
zearalenone and its analogs, along with several other mycotoxins (such as
deoxynivalenol, nivalenol and fumonisins), have been detected as secondary
metabolites of Fusarium species in many mold contaminated grains and fruits,
including corn, wheat, barley, oats, sorghum, sesame (Mirocha et al., 1977a),
soybean (Shotwell et al., 1977), cottonseed, peanut (Price et al., 1993), walnut
(Jemmali and Mazerand, 1980), rice, rapeseed (Chakrabarti and Ghosal, 1987),

banana (Chakrabarti and Ghosal, 1986), tomato (El-Morshedy and Aziz, 1995),

OH
OH 0 Me
00".
H0 o H

Zearalenone Estradiol

OH 0 Me OH 0 Me
Ho OH H' OH

a—Zearalenol B-Zearalenol

OH 0 Me OH 0 Me
m
H “OH H' OH

a—Zearalanol (Zeranol) B-Zearalanol

Figure1.1. Structures of zearalenone and analogues showing structural
similarities with estradiol.

 

 

 

 

 

I\l

beets (Bosch and Mirocha, 1992), and amaranth grain (Bresler et al., 1991 ). Corn
is the most common grain contaminated with zearalenone and its derivatives
(Wood, 1992). The occurrence of zearalenone ranged from 0.1 to 8 ppm in corn
samples from several surveys in the United States (Stahr et al., 1981; Cote et al.,
1984; Abbas et al., 1986; Hart, 1987).

Normally, zearalenone production is promoted by high humidity and low
temperatures, as is common in the upper Midwest during autumn harvest
(Coulombe, 1993). Zearalenone production is also correlated with plant density, soil
fertility, insect damage, mechanical damage during harvest, and improper storage
conditions (Mirocha et al., 1977a; Sutton et al., 1980; Montani et al., 1988).

Zearalenone, in spite of its large lactone ring, is surprisingly stable to
hydrolytic cleavage (Mirocha et al., 19773). It is also relatively stable during the
processing of feed and foods. Zearalenone and its analogs enter the food chain
directly through contaminated grain-based food and animal feed. Once zearalenone
and its derivatives are produced in grains, they will remain in the grain-based feed
or food. In two separate surveys of processed grain-based products (breakfast
cereals, popcorn, snack foods, corn meal, crackers/cookies/muffin mixes)
conducted during 1985 and 1989, the same relative concentrations of zearalenone
(average 19 to 20 uglg )were detected (Warner and Pestka, 1987; Abouzied et al.,
1991 ). Additionally, the use of zeranol (Ralgro)(a-zearalanol) (a synthetic derivative
of zearalenone) (Figure 1.1) as a growth-promoting agent in beef cattle and sheep
provides another route to enter the food chain (Sundlof and Strickland, 1986).

Zeranol has been found in the edible tissues (such as muscle, liver, kidney, and fat)

of the animals implanted with this growth-promoting agent (Dixon and Russell,
1986)

Distribution, elimination and metabolism. When ingested orally by
animals, zearalenone appears to be fairly rapidly absorbed through the gut into the
circulatory system (Dailey et al., 1980; Olsen et al., 1985a). Zearalenone can then
be transported to various organs and tissues. The liver appears to be the main
organ of deposition upon absorption (Chichila et al., 1988). Zearalenone is also
distributed into adipose tissue, ovary, utenls, testicle, and other sites (Ueno et al.,
1977; Appelgren et al., 1982).

Zearalenone can be rapidly eliminated from an animal. Following a single
oral dose of zearalenone in rats, about 90% of the dose was excreted in feces after
48 hr, while the remaining 10% was excreted in urine (Smith, 1982). Zearalenone
and metabolites were excreted mainly in the free form with conjugates being found
only in urine. Using different oral doses of zearalenone in rats, Fitzpatrick et al.
(1988) found that dose had little effect on metabolites formed, or excretion route.
In one study, Pandey et al. (1990) showed that UDP-glucuronyltransferase induction
by phenol barbital increased urinary excretion of conjugated a—zearalenol. A more
recent study with administration of [3H] zearalenone showed that the biological
half-life of zearalenone in plasma was 86 hr for intravenously or orally dosed
immature pigs, while only 3.3 hr for intravenously dosed animals after bile removal
(Biehl et al., 1993). It provided evidence for extensive biliary secretion and
enterohepatic cycling of zearalenone and metabolites in pigs. Besides the route of

feces and urine, a small portion of zearalenone can be excreted into milk as

 

 

 

 

 

al.

an

SBJ

vul
Ch

mo

Oil

Ani

re pl

(Mil

conjugates (Prelusky et al., 1990).

Zearalenone is metabolized mainly in the liver through dehydrogenation and
glucuronide conjugation (Kiessling and Pettersen, 1978; Mirocha et al., 1981 ; Olsen
et al., 1981; Bock et al., 1987). Considerable differences between species exist in
the ability to reduce zearalenone (Olsen and Kiessling, 1983; Pompa et al., 1986).
Among five species examined, swine had one of the greatest abilities to form the
more highly estrogenic a-zearalenol (Olsen et al., 1985b). In some cases, sulfo-
conjugates of zearalenone and its analogs were also found at low levels (Olsen et
al., 1986; Bories et al., 1991).

Toxicity. With their chemical similarity to estradiol (Figure 1 .1 ), zearalenone
and its derivatives bind to mammalian estrogen receptors in reproductive and other
sensitive tissues (Hurd, 1977). When fed to animals, zearalenone causes
hyperestrogenism with symptoms such as enlargement of the uterus and nipples,
vulvar swelling, vaginal prolapse, and infertility (Hidy et al., 1977; Mirocha and
Christensen, 1974). Swine are the most sensitive of the large domestic animals and
most commonly affected with hyperestrogenism on the farm although bovine
hyperestrogenism due to zearalenone is also suspected (Mirocha et al., 1977b).
Other farm animals affected by zearalenone are dairy cattle, chickens and turkeys.
Animals experimentally affected are rats, mice, guinea pigs, monkeys and lambs
(Mirocha et al., 1977). Embryotoxicity and teratogenic effects have also been
reported in some animals after consumption of zearalenone-contaminated feeds
(Miller et al., 1973; Sharma et al., 1974; Shreeve et al., 1978; Chang et al., 1979;

Becci et al., 1982). Zearalenone and its derivatives also affect the immune system

10

 

 

 

 

 

 

anabo

probe,
zearal

Sheep

of animals. They are capable of inhibiting mitogen-stimulated lymphocyte
proliferation (Forsell and Pestka, 1985). Dietary exposure to 10 ppm of zearalenone
for two weeks decreases the resistance of mice to Listeria monocytogenes (Pestka
et al., 1987). Zeranol could induce acute hepatotoxicity and, subsequently, hepatic
carcinogenesis in Armenian hamster (Coe et al., 1992). More recently, DNA adducts
were also found in the kidney, liver and ovary of female mice treated with
zearalenone (Pfohl et al., 1995; Grosse et al., 1997).

Using the closely related a-zearal'enol, the no-honnonal effect level for
zearalenone was estimated to be 50-60 ug/kg body weight per day, based on a 90-
day oral study in ovariectomized monkeys with vaginal comification as the endpoint
(Griffin et al., 1984; Lindsay, 1985; Kuiper-Goodman et al., 1987). Dividing the non-
honnonal effect level by a safety factor of 500, which takes into account inter— and
intraspecies differences and uncertainties in the experiment model, Kuiper-
Goodman et al. (1987) gave an estimated Tolerable Daily Intake (T DI) of 100 ng/kg
body weight per day for humans.

Although zearalenone and its derivatives are toxic at high doses, they act as
anabolic hormones enhancing growth when used at the proper time and in the
proper amounts. Notably, zeranol (Ralgro) (a-zearalanol), a synthetic derivative of
zearalenone, has been approved as a growth-promoting agent in beef cattle and

sheep (Umberger, 1975).

11

 

 

Fumon

produce
F. proif
sorghui
F. moni
causati
1904). '

leukoer

 

charact
hemiso

obsew

Cher
199
DTOC
an 6
al., 1

Cam;

Fumonlsins

Historical background. Fumonisins are a group of secondary metabolites
produced by Fusarium moniliforme (Sheeldon) (Benzuidenhout et al., 1988) and
F. proliferatum (Ross et al.,1990). These fungi are commonly found on corn,
sorghum, and other grain commodities throughout the world (Marasas et al., 1984).
F. moniliforme, which was firstly described by Sheldon (1904), was implicated as a
causative agent of “moldy corn poisoning” in animals in the United States (Peter,
1904). This mold has been associated with an equine poisoning known as equine
leukoencephalomalacia (ELEM) (Ross, 1994). This is a neurotoxicosis
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
B1 (FB,)] 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).

Six different fumonisins, fumonisin A,, A2, B1, B2, Ba, and B,, have been
chemically characterized (Figure 1.2) (Bezuidenhout et al., 1988; Cawood et al.,
1991; Plattner et al., 1992). FB1 is the most toxic and predominant fumonisin
produced by F. moniliforme (Gelderblom et al., 1988a), and has been confirmed as
an etiologic agent of fatal animal diseases such as ELEM in horses (Kellerrnan et
al., 1990), porcine pulmonary edema (PEP) in pigs (Harrison et al., 1990), and liver

cancer in rats ( Gelderbolm et al., 1991). In addition, epidemiological studies

12

 

 

 

 

 

inc

3C

pe

be
de

of

Bi

all

WI'

be

indicate that fumonisins are associated with human esophageal cancer in South
Africa (Marasas et al., 1988a).

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 deoxy analogue (Figure 1 .2) (Bezuidenhout et al.,
1988). Chemical structures of these toxins are similar to that of Altemaria altemata
f. sp. lycopersici (AAL) toxin and sphingosine (Figure 1.2) (Norred, 1993).

The biosynthetic pathways through which fungi produce fumonisins have only
been partially elucidated (Norred, 1993). When Plattner and Shackelford (1992) fed
deuterium-labeled methionine to F. moniliforme cultures, they observed high levels
of deuterium incorporation in FB1 at the methyl groups on the C-12 and C-16
position of fumonisin backbone. Based on 13C-labeled acetate feeding experiment,
Blackwell et al. (1994) concluded that the backbone of fumonisins was synthesized
by the fungus through condensation of acetyl coenzyme A and serine. However,
more work is still required to fully elaborate the biosynthetic pathway of the
fumonisins.

Natural occurrence. F. moniliforme, the fumonisin-producing fungus,
commonly occurs in the world and it has been isolated in many countries (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 cat and other agricultural products such as beans, peanuts, sugar

beats, and bananas (Bacon and Nelson, 1994).

13

FUMONISINS

 

     

 

R1 R2 R3 COOH
FAl OH OH CH2CO cocnzICchzcoon
FA2 H OH CH2CO I R, 0,,
F81 OH OH H 20 1CH3
F82 H OH H
F83 OH H H 21 CH3 0 22 H3 1 NHR3
FB4 H H H COCHzCHCHzCOOH
OOH
COOH
cocr—rzcncnzcoon
OH on
AAL TOXIN W
CH3 on CH3 OH NHz

OH

SPHINOSINE chzm

NH2

Figure 1.2. Structures of known fumonisins and Altemaria altemata f. sp.
chopersici (AAL) toxin, showing structural similarities with
sphingosine (Norred, 1993).

14

 

 

 

 

 

C

 

storage
reporter
aerated
product
FB, at It
conditi

1990).

caused
Maras

the hor
oi the I
ELEM
body \
Sufien

ELEM

Edem,
alooj

et at,

bIOSYn

 

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, 32% moisture content of corn media, and
aerated atmosphere (cotton-stoppered flasks) were the optimum condition of FB1
production by F. moniliforme in laboratory experiments. F. moniliforme can produce
F B1 at level of 300 to 17,000 ppm when cultured on solid corn media at the optimum
conditions for 2 to 12 weeks (Bars et al., 1994, Nelson et al., 1991, Alberts et al.,
1990)

Toxicity. Equine leukoencephalomalacia (ELEM) is the most studied disease
caused by consumption of F. moniliforme contaminated corn (Norred, 1993;
Marasas et al., 1988b). ELEM develops over a several-week period, during which
the horse may refuse feed, becomes lethargic and is disoriented. The progression
of the condition includes ataxia, profuse sweating, convulsions, and finally death.
ELEM has been reproduced by intravenous administration of 7 doses (0.125mg/kg
body weight) of purified FB1 over 10 days (Marasas et al., 1988b). The horses
suffered convulsions and were found to have brain lesions similar to those seen in
ELEM cases (Merrill et al., 1995).

Porcine pulmonary edema (PPE) which develops hydrothorax and lung
edema usually resulting in death was particulariy prevalent in the United States
when pigs were fed contaminated corn (Norred, 1993). Pigs that received pure FB1
at 0.4 mg/kg body weight were dead on day 5, with typical PPE lesions (Harrison
et al., 1990). Haschek et al. (1992) suggested that the disruption of sphingolipid

biosynthesis by FB1 causes membranes damage in hepatocytes and release into

15

the blood stream. Pumonary intravascular macrophages (PlMs) which phagocytize
the membrane fragments are especially rich in the lungs of pigs. This initiates the
PlMs to release enzymes and other mediators, which increase capillary permeability
in the lung tissue and lead to edema (Haschek et al., 1992; Merril et al., 1995).

In addition, fumonisins can cause nephrotoxicity, hepatotoxicity and
hepatocarcinogenicity in some experimental animals (Jaskiewicz et al., 1987;
Gelderblom et al., 1991; 1992; Riley et al., 1994; Voss et al., 1995). More
importantly, human esophageal cancer in the areas of Southern Africa and China
has been correlated with the consumption of corn contaminated by F. moniliforme

(Marasas et al., 1988a; Marasas, 1995; Cheng et al., 1985; Chu and Li, 1994).

Trichothecenes

Occurrence. Trichothecenes are a group of structurally similar
sesquiterpenoid metabolites (Figure 1.3) produced mainly by Fusarium spp.
(Bamburg and Strong, 1971 ; Bamburg, 1983; Ueno, 1983; Betina, 1989a) . The first
known member of this group, trichothecin, was originally discovered as an
antifungal antibiotic in 1948 (Freeman and Morrison, 1948; 1949). Since that time
over 80 trichothecenes have been isolated and characterized (Betina, 1989b). But
only a few members of the trichothecenes, such as deoxynivalenol (DON,
vomitoxin), nivalenol (NIV), T-2 toxin, HT-2 toxin, and diacetoxyscirpenol (DAS) are

detected as major natural contaminants in cereal grains (Betina, 1989b).

16

 

 

 

 

 

 

 

----R

3 1

4t”-H

R2
Trichothecene R1 R2 R3 R4 R5
T—2 toxin OH OAC OAC H OOCCH2CH(CH3)2
HT-2 toxin OH OH OAC H OOCCH2CH(CH3)2
T-2 tetraol OH OH OH H OH
Diacetoxscirenol OH OAC OAC H H
Scirpenetriol OH OH OH H H
Deoxynivalenol OH H OH OH O
Nivalenol OH OH OH OH O
Fusarenon-X OH OAC OH OH O

 

Figure1.3. Structure and numbering system of some naturally identified
trichothecene mycotoxins (modified from Ueno, 1983).

17

 

 

 

enviro
Wet a
trichc
Uenc
of de

Sch

red

SUI

Production of trichothecenes in cereal grains is primarily dictated by
environmental conditions (Vesonder et al., 1978; Ueno, 1983; Cote et al., 1984).
Wet and cool seasons in the spring and early summer favor Fusarium infection and
trichothecene production in the field (T uite et al., 1974; Shotwell et al., 1977; 1983;
Ueno, 1983; CAST, 1989). In a year when wheat scab is epidemic, the occurrence
of deoxynivalenol could be detected up to12 ppm in bulk grain samples (Hart and
Schabenberger, 1998).

Toxicity. Symptoms in acute experimental trichothecene poisoning are
reduced weight gain, anorexia, dermal irritation, abortion and hematological lesions
such as leukopenia, anemia and lack of bone marrow development (Ueno, 1987;
Rotter et al., 1996). Major human outbreaks of trichothecene toxicoses have been
reported in Russia, Japan, China and India (Beardall and Miller, 1994).
Consumption of trichothecene—contaminated grains causes headaches, chills,
severe nausea and vomiting, diarrhea, visual disturbances and fatal hemorrhage in
humans (Watson et al., 1984). Trichothecenes also modulate immune function and
their ingestion over prolonged periods may reduce the immune resistance to
infectious diseases, facilitate tumor growth by impairing immune surveillance
(Pestka and Bondy, 1994), and cause autoimmune disease, as demonstrated in
experimental deoxynivalenol-induced IgA glomerulonephritis (Dong et al., 1991;

Dong and Pestka, 1993; Rasooly et al., 1994; Rasooly and Pestka, 1994).

18

Analytical methods for mycotoxin detection

The presence of mycotoxins in foods and feeds is potentially hazardous to
the health of humans and animals. Although it is difficult to remove mycotoxins from
human and animal diets, it is possible to decrease the risk of exposure through a
rigorous program for monitoring these toxins in foods and feeds and diversion of
the contaminated materials (Chu, 1992; Pestka et al., 1995). In general, analysis
of mycotoxins in food and feed is a difficult task because only trace amounts of the
toxins are present in the sample. The analysis is further complicated by the uneven
distribution of mycotoxins in the sample and by sample matrix interference.
Normally, the suspected contaminated commodity is first subjected to a rigorous
sampling program (Campbell et al., 1986). The toxin is then extracted from the
sample and subjected to a very extensive cleanup treatment to remove interfering
substances before analysis is conducted. Current analytical methods for mycotoxins
include thin layer chromatography (TLC), high performance liquid chromatography
(HPLC), gas chromatography (GC), mass spectroscopy (MS), immunoassay and
bioassay.

For mycotoxins that contain a chromophore group with fluorescent
properties, such as zearalenone, sample extracts after cleanup are generally
subjected either to TLC or to HPLC to separate different structurally related
compounds and contaminants (Chu, 1992; Richard et al., 1993). Identification of the
toxin is made by comparing the retention factor (Rf) values or retention time in the
chromatogram with the standard, whereas quantitative information is obtained from

fluorescence intensity or absorbance of the elution peak (or spot). Some

19

mycotoxins, such as trichothecenes and fumonisins, do not have well-defined
absorption maxima or potential for fluorescence under ultraviolet or visible light.
Therefore, derivatization reagents are required to provide a fluorescent
characteristic for their detection (Chu, 1992; Richard et al., 1993). An effective TLC
or HPLC method can detect zearalenone, fumonisins and deoxynivalenol at 50 to
300 ppb (Richard et al., 1993). Higher sensitivity and selectivity of mycotoxin
analysis can be achieved by using GC and GC/MS techniques although they require
laborious sample cleanups, and expensive instruments (Pestka, 1988, 1994; Chu,
1991).

Immunoassays have been widely used for mycotoxin analysis at the pg and
ng levels (Pestka et al., 1995). The most common immunoassay, enzyme-linked
immunosorbent assay (ELISA), involves competition between a free mycotoxin in
a sample extract with an enzyme-labeled mycotoxin for an antibody binding site.
Mycotoxins can be analyzed directly in a liquid system such as milk and solid
substrates can be extracted with methanol-water and analyzed directly or after
dilution. Because mycotoxins alone are not antigenic, early studies focused on the
development of methods for conjugation of mycotoxins to a protein or polypeptide
carrier and optimization of conditions for antibody production in rabbits and in other
animals (polyclonal antibody). With the advances in hybridoma technology,
monoclonal antibodies against many important mycotoxins, including zearalenone,
fumonisin B,, and deoxynivalenol, were also generated (Pestka et al., 1995).

Compared with other methods, mycotoxin immunoassays have several advantages

20

for rapid field tests, including high specificity, sensitivity, facile sample preparation,

and ease of use (Pestka et al., 1995).

21

RECOMBINANT ANTIBODIES AND PEPTIDE MIMOTOPES

The wide application of mycotoxin immunoassay has led to a great demand
for immunochemical reagents including low-cost antibodies with high affinity and
defined specificity. With the advances in the field of recombinant antibody
technology, it is now theoretically feasible to engineer low cost antibodies for
mycotoxins with desirable affinity and specificity characteristics by using
recombinant DNA technology to manipulate the basic domain structure of the
immunoglobulin molecule and to express it in bacteria (Hoogenboom et al., 1992;
Lerner et al., 1992). Peptide mimotopes to mycotoxins may also be selected from

a random peptide library and used as alternative immunochemical reagents.

Native antibody

An immunoglobulin (lg) molecule is composed of two heavy (H) and two light
(L) protein chains (Figure 1.4). These protein chains contain defined Variable (V),
Diversity (D, heavy chain only), Joining (J) and Constant (C) regions. The variable
region is positioned at the amino-terminus and is composed of alternating
framework (Fw) and hypervariable or complemetarity—determining region (CDRs).
The VL and VH regions constitute the basic antibody binding site (paratope) which
is dictated by the CDRs and, to a lesser extent, the Fw region. This domain
structure encoded by variability (V) genes facilitates the extensive gene
rearrangement that occur in B cells during in vivo immune selection. When newly

generated B cells bind antigens via the lg receptor, they differentiate (with T cell

22

help) to either short-lived lg secreting plasma cells or long-lived memory cells that
are involved in secondary/tertiary responses. During the selection process, highly
frequent somatic mutations in the rearranged lg genes of the memory B cells

greatly improve antibody affinity.

Recombinant antibody and phage display

Recombinant antibody technology involves rearrangement or assembly of
antibody V-genes, surface display of antibody, affinity selection, affinity maturation
and soluble antibody production (Winter at 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). But screening specific antibody from large combinatorial repertoires was
a difficult task.

In 1985, a report was published describing the in-frame insertion of a DNA
fragment encoding a section of the restriction endonuclease (5le into the gene of
a minor capsid protein (93p) of the filamentous bacteriophage fd (Smith, 1985). The
filamentous fd bacteriophage is male specific, containing circular single-strand DNA
(Model and Russel, 1988) . The phage genome (approximately 6500 nucleotides
in length) encodes 10 proteins (Table 1.1). An important feature of filamentous fd
phage is their ability to package longer genomes than the wild-type DNA, simply by
further addition of 98p subunits. Fusion phages have been generated with insertions

into either the minor capsid protein g3p for large insertions, or the major capsid

23

Figure 1.4.

 

Antibody model structures showing subunit composition and domain
distribution along the polypeptide chains. Fragments generated by
proteolytic cleavage and /or recombinant technology appear in the
shaded area (Phannacia Biotech).

24

Table 1.1. The genes and proteins of fd bacteriophage

 

 

 

 

 

 

 

 

 

 

 

Gene Protein size Copy number Protein function

1 35-40 KDa few/cell NS membrane protein; interacts with host
thioredoxin; required for assembly.

2 46KDa ~103/cell Site- and (+) strand-specific endonuclease!
topoisomerase; required for replication of RF.

X 12 KDa ~500/cell N-terminal fragment of gene 2; required for
replication of RF.

3 42 KDa 5/particle At one end of particle; required for correct
morphogenesis of unit-length particles; N-terminal
part binds to F pilus of host cell.

4 49KDa few/cell NS membrane protein; required for assembly.

5 10KDa ~105lcell Major structural protein during replication; controls
expression of 92p; binds to DNA; replaced by g8p
during assembly; controls switch from RF
replication to progeny (+) strand synthesis.

6 12KDa ~5/particle At the same end of particle as g3p; involved in
attachment and morphogenesis.

7/9 3.5KDa ~5 of each At opposite end of particle to gSp/g6p; involved in

[particle assembly;

8 5KDa 2,700-3,000/ Major coat protein.

particle

 

 

 

 

25

 

protein g8p for short peptide insertions (Smith, 1993). In these “fusion phages’, the
DNA encoding the insert protein is packaged as an integral part of the phage
genome, while the chimeric protein product is expressed on the surface of the
bacteriophage particle, allowing the simultaneous selection of a gene and its
protein. Since 1985, this ingenious system linking both the DNA Sequence and its
expression product in a single phage particle has resulted in a rapidly expanding
technology with diverse applications (O’Neil and Hoess, 1995; Hill and Stockley,
1996)

A rapidly expanding application of phage display technology has stemmed
from the discovery that antibody fragments can be presented on the surface of '
bacteriophage in a biologically active form (McCafferty et al., 1990) and the
repertoires of heavy- and light-chain variable region genes can be built by using
polymerase chain reaction (PCR) (Orlandi et al., 1989). In the last decade, many
antibody or antibody fragment genes have been cloned with phage display.
Antibody phage display is accomplished by fusing the coding sequence of the
antibody variable (V) regions to the amino terminus of the phage minor coat protein
93p (McCafferty et al., 1990). Expression of the fusion product and its subsequent
incorporation into the mature phage coat results in the antibody being presented on
the phage surface, while its genetic material resides within the phage particle. This
linkage between antibody genotype and phenotype allows the enrichment of
antigen-specific phage antibodies, using immobilized or labeled antigen. In a
screening procedure, called “biopanning” (Marks et al., 1991), phage that display

a relevant antibody will be retained on the surface coated with antigen, while non-

26

adherent phage will be washed away. Bound phages can be recovered from the
surface, re-infected into bacteria and re-grown for further enrichment and,
eventually, for binding analysis (Marks et al., 1991; Hawkins et al., 1992; Yuan et
aL,1997)

Affinity and specificity in phage-displayed antibodies can be further enhanced
by mimicking B cell somatic mutation via in vitro mutagenesis or by shuffling a
repertoire of in vivo somatically mutated V-genes (Hoogenboom et al., 1992). The
CDR regions are often the targets for antibody in vitro mutation and maturation.
Using “CDR-walking”, Yang et al. (1995) created independent libraries with
mutations in one of four CDR regions, combined the selected variants in one clone,
and obtained a 420-fold increase in affinity. The CDR3 of the heavy chain was
demonstrated to be most important for in vitro affinity maturation (Thompson et al.,
1996; Schier et al., 1996; Yang et al., 1995).

Chain shuffling is the sequential replacement of VH and VL region genes with
repertoires of V-genes (Marks et al., 1992a). Chain shuffling of V-genes has been
used successfully to obtain a 300-fold increase in the affinity of an anti-hapten
antibody, originally isolated from a naive human scFv library (Marks et al., 1992b).
The source of the V-genes for chain shuffling can be a library of light or heavy chain
V-genes from non-immunized animals (naive library) (Marks et al., 19923), from
animals immunized with an antigen of interest (Kang et al., 1991) , or from other
positive clones in the initial library (Clackson et al., 1991).

Several types of antibody constmct have been displayed including the

variable region of single heavy chains, and single-chain Fv fragments (scFv) (Hill

27

and Stockley, 1996). Fab fragments have also been displayed in a form in which
either the light or heavy chain is expressed as a fusion on the bacteriophage
surface with the complementary heavy or light chain associated via its secretion into
the periplasm of phage-infected cells (Barbas et al., 1991; Hoogenboom et al.,
1991)

Compared to Fab and Fv fragments, single-chain Fv fragments fold readily
in the correct conformation and have improved stability because the variable heavy
(VH) and variable light chain (VL) domains are covalently joined by a polypeptide
linker (Raag and Whitlow, 1995). Generally, peptide sequences from known protein
structures are used as linkers, whose lengths and conformations are compatible
with bridging the variable domains of an scFv without serious steric interference
(Bird et al., 1988; Takkinen et al., 1991; Mallender and Voss, 1994). The most
widely used linker designs have sequences consisting primarily of stretches of Gly
and Ser residues for flexibility, sometimes with charged residues such as Glu and
Lys interspersed for solubility (Bird et al., 1988; Huston et al., 1988; Whitlow et al.,
1993).

The stability of a particular scFv form depends on a number of factors, in
addition to the nature of the V,,,/VL interface. These include the presence of antigen,
the length of the linker, the solvent system, and temperature. Longer linkers were
found to stabilize scFv antibodies (Whitlow et al., 1993; Holliger et al., 1993), while
some organic solvents, such as ethylene glycol (Kortt et al., 1994), ethanol (Essig
et al., 1993), and methanol (Yuan et al., 1997) were showed to destabilize scFvs.

Some strategies generally applicable to protein stabilization may be used to

28

stabilize the scFv (Raag and Whitlow, 1995). These include the engineering of
stronger hydrogen bonding interaction (Pantoliano et al., 1989), disulfide bonds
(Glockshuber et al., 1990; Btinkmann et al., 1993; Luo et al., 1995; McCartney et
al., 1995), metal binding sites (Wade et al., 1993), increasing buried hydrophobic
areas, and adding or improving electrostatic interactions (Jones et al., 1992).
The majority of scFv applications involve a bifunctional task in which scFv
binding to a target antigen allows high specific delivery of a second effector function
(Raag and Whitlow, 1995). They might also be used as diagnostic reagents.
Antibody fragments, specific for an important pollutant or a toxicant, could have
considerable potential for the detection and removal of trace levels of contaminants.
For example, a cloned antibody fragment to the herbicide paraquat has been shown
to be capable of removing paraquat from aqueous environments (Graham et al.,
1995). Antibody fragments with catalytic activities have also been developed using
phage display (McCafferty et al., 1994; Janda et al., 1994; Baca et al., 1997).

Peptide mimotopes

The physical linkage between a vast library of variants with various binding
activities to genes encoding each variant from phage display also permits the
identification of peptides with certain binding functions. In 1990, three groups
simultaneously reported the construction and screening of highly diverse libraries
of unstructured random peptides (6-12 residues) displayed on fd phage or the
related phage M13 as g3p fusions (Scott and Smith, 1990; Cwirla et al., 1990;

Devlin et al., 1990). In a complementary strategy, peptide libraries fused to the

29

major coat protein, g8p, have also been used for epitope mapping (Felici et al.,
1991). The increased valency of 98p fusions permits selection of lower-affinity
ligands, while g3p library generally produce higher affinity ligands.

Peptide libraries can be structurally constrained by flanking the randomized
segment with a pair of cysteines which form a disulfide bond (McLafferty et al.,
1993; McConnell et al., 1994; Giebel et al., 1995). Cycllzation can mimic the native
structural context of epitope sequences by ‘freezing out” unproductive
conformations. Structurally-constrained peptide libraries are valuable in identifying
structural epitopes (Hoess et al., 1994).

In the last 8 years, random peptide libraries displayed on phage have been
extensively used to select for peptide ligands specific for a variety of targets
(antibodies, receptors, enzymes, etc.) by using multiple cycles of in vitro selection
(biopanning) and in vivo amplification (Parrnley and Smith, 1988; Smith and Scott,
1993). Phage-display libraries have also been used to identify peptide mimics of
non-peptide ligands (Devlin et al., 1990; Scott et al., 1992; Oldenburg et al., 1992;
Hoess et al., 1993; Adey et al., 1995).

30

PLANTIBODIES

Antibodies historically have been considered as part of mammalian defense
systems. With the advances in antibody gene cloning and plant genetic
transformation, however, the ability to produce antibodies and antibody fragments
in plants presents some very interesting possibilities. These so-called “plantibodies”
can be produced on an agricultural scale for therapeutic or diagnostic use (Smith
1996). They also provide the opportunity to manipulate plant traits or reduce
pathogenic infections in plants through antibody-mediated modifications of antigen
activity in planta (Hiatt, 1990; Schots et al., 1992; Tavladoraki et al., 1993; Voss et
al., 1995; Whitelam, 1995). The binding and retention capacity of antibodies could
also be used for the isolation and processing of environmental contaminants, a
process known as biofiltration (Hiatt, 1990).

For expression of complete antibodies or Fab fragments, in one approach,
light- and heavy-chain genes were separately introduced into plants. Plants
expressing light and heavy chains were then crossed and assembled antibodies
were detected in the F1 generation (Hiatt et al., 1989; Ma et al., 1994; Ma et al.,
1995). Alternatively, double transformations were performed or the genes for
expressing light and heavy chains were cloned into one expression cassette (De
Neve et al., 1993; Dilring et al., 1990; Voss et al., 1995). The expression of a
functional scFv antibody in plants is relatively simple because it requires only

correct folding and it does not have to be assembled like complete antibodies.

31

Antibodies have been successfully expressed in plants both intracellulariy
and intercellulariy. Full length antibodies in algal cytoplasm can form correctly folded
antigenic binding sites even though the relatively high reducing conditions in the
cytoplasm do not allow formation of disulfide bonds (Stieger et al., 1991). However,
functional full length antibodies in the cytoplasm have not yet been reported in
higher plants. It is possible that the cytoplasm of algae is less reducing and contains
the chaperonins necessary for folding of full-size immunoglobulins (Smith, 1996).
In contrast, the expression of scFv antibodies in the plant cytoplasm has been more
successful. Some groups have reported successful expression of scFv antibodies
in the cytoplasm (Owen et al., 1992; Tavladoraki et al., 1993), although others failed
to get any antibody accumulation unless an endoplasmic reticulum (ER) retention
signal (a tetrapeptide KDEL) was included (Schouten et al., 1996).

The production of secreted antibodies in plants normally requires a signal
sequence, although the origin of the signal sequence does not appear to be critical,
since plant, mouse, and yeast sequences can be used (DlJring et al., 1990; Hein et
al., 1991). The signal sequences not only direct the antibody fragments to plant
secretory pathways but also stabilize the antibody and increase the yield (Hiatt et
al., 1989; Firek et al., 1993). Although plant cell walls are believed to have exclusion
limits of between 17 and 60 KDa (Carpita et al., 1979; Tepfer and Taylor, 1981; De
Wilde et al., 1996), the results of De Wilde et al. (1996) suggest that the mesophyll
cell walls of Arabidopsis are permeable to intact IgG molecules, which have a
molecular weight of 146 KDa. These findings are of particular interest to any

application where extracellular antigen binding is desired. When a suitable signal

32

sequence was used, antibody was also targeted into plant nuclei (De Neve et al.,
1993).

Antibody can also be targeted to specific plant organs. Fiedler and Conrad
(1995) directed active scFv molecules into tobacco seeds and stored them stably
for one year at room temperature. Such a system would be valuable for long-term
storage and easy delivery of large quantities of antibodies for passive immunization.

As in mammals, y—heavy chain expressed in plant has N-linked glycosylation,
in which a high-mannose core oligosaccharide is attached to the asparagines
contained within the canonical Asn-X-Ser/Thr sequence (Hein et al., 1991).
However, the composition of terminal residues on the plant glycan appears to be
distinct from that in mammals. The differences in glycosylation patterns of plant
antibodies had no effect on antigen binding or specificity (Hein et al., 1991).
However, for human therapy, the presence of plant-specific glycan might increase
the immunogenicity of the recombinant antibody.

Since the first plantibody was expressed in tobacco (Hiatt et al., 1989), a
number of antibodies or antibody fragments have been successfully produced in
plants for various purposes (Table 1 .2). Full length antibodies have been expressed
in plants with the aim of producing large quantities for use in immunotherapy.
Indeed, the production of multimeric secretory antibodies (Ma et al., 1995) and the
ability to store antibodies long-term in seeds or other organs (Fiedler and Conrad,
1995) showed great promise in immunotherapy and passive immunization by using
plantibody. In an effort to confer viral resistance to plants, Tavladoraki et al. (1993)

expressed an scFv antibody against the artichoke mottled crinkle vims in the

33

h\

In

A.

cytoplasm of tobacco. These transgenic plants were specifically protected from viral
attack, in that they had a reduced incidence of infection and showed delayed
symptom development ( Tavladoraki et al., 1993). The possibility of using full length
antibodies to interfere with fungal pathogenesis in the apoplast has also been
examined (van Engelen et al., 1994). With the intracellular expression of specific
antibody fragments in plants, plant physiological traits such as phytochrome-
mediated seed germination (Owen et al., 1992) and abscisic acid-controlled
physiology (Artsaenko et al., 1995; Phillips et al., 1997), have successfully been
altered.

The feasibility of producing specific antibody or antibody fragments in plants
allows us to speculate that the expression of mycotoxin specific antibody in plants
may be useful to reduce plant and animal diseases associated with the mycotoxin

by antibody-mediated neutralization and degradation.

Table 1.2. Antibody—derived molecules produced in transgenic plants

 

 

 

Antibody form Antigen Plant species Reference
Single domain Substance P Nicotiana Benvenuto et al., 1991
(sAb) (neuropeptide)
Single chain Fv Phytochrome Nicotiana Firek et al., 1993;
(scFv) Owen et al., 1992
Single chain Fv Artichoke mottled crinkle Nicotiana Tavladoraki et al., 1993
(scFv) virus coat protein
Single chain Fv Beet necrotic yellow vein Nicotiana Fecker et al., 1996
(scFv) virus coat protein
Single chain Fv Abscisic acid Nicotiana Artsaenko et al., 1995
(scFv) Phillips et al., 1997
Fab; IgG (1:) Human creatine kinase Nicotiana De Neve et al., 1993
Arabidopsis
IgG (1:) Transition-state analogue Nicotiana Hiatt et al., 1989;
Hein et al., 1991

lgG (x) Fungal cutinase Nicotiana van Engelen et al., 1994
lgG (x/k) TMV Nicotiana Voss et al., 1995
lgG (x) and lgG/A Streptococcus mutants Nicotiana Ma et al., 1994
hybrids adhesin
Secretory lgA/G S. mutants adhesin Nicotiana Ma et al., 1995
IgM (k) NP (4-hydroxy-3- Nicotiana Dtlring et al., 1990

nitrophenyl) acetyl hapten
lgG-like lgM Nematode stylet Nicotiana Baum et al., 1996

secretions Rosso et al., 1996

 

35

 

PART II
MOLECULAR CLONING, EXPRESSION AND CHARACTERIZATION OF A

FUNCTIONAL SINGLE-CHAIN Fv (scFv) ANTIBODY
TO THE MYCOTOXIN ZEARALENONE

36

ABSTRACT

MOLECULAR CLONING, EXPRESSION AND CHARACTERIZATION OF A
FUNCTIONAL SINGLE-CHAIN Fv (scFv) ANTIBODY
TO THE MYCOTOXIN ZEARALENONE

The heavy and kappa light-chain variable region genes of an anti-
zearalenone hybridoma cell line (2GB—6E3-2E2) were isolated by polymerase chain
reaction (PCR) and joined by a DNA fragment encoding the linker peptide (Gly.,Ser)3
to generate a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was
cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with an
antibody recognition sequence (E-tag) and phage M13 coat protein 93p in
Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion
protein was displayed on the surface of recombinant phage. High affinity scFv
phage antibodies were enriched through panning-elution selection in microtiter wells
coated with zearalenone-ovalbumin conjugate. The selected recombinant phages
were used to infect E. coli HB2151 for the production of soluble scFv antibodies.
One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5)
with high zearalenone binding affinity (IC50=14 ng/ml), similar to that of the parent
monoclonal antibody in a competitive indirect ELISA. However, scFv QY1.5
exhibited higher cross-reactivity with zearalenone analogues and had increased
sensitivity to methanol destabilization when compared with the parent monoclonal
antibody. Nucleotide sequence analyses revealed that the light chain portion of

scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse gerrnline gene

37

VK23.32 in mouse kappa variable light chain subgroup V, whereas the heavy chain
nucleotide sequence was classified as mouse heavy chain subgroup Ill (D) but
without any closely related members having highly homologous complementary-
deterrnining regions (CDRs) sequences. The potential of soluble scFv QY1.5 for
routine screening of zearalenone and its analogues was demonstrated with
zearalenone spiked corn extracts. A portion of the research presented here has
been published (Yuan, 0., Clarke, J. R., Zhou, H. R., Linz, J. E., Pestka, J. J., and
Hart, L. P. 1997. Molecular cloning, expression and characterization of a functional

single-chain Fv antibody to the mycotoxin zearalenone. Appl. Environ. Microbiol.

63:263-269).

38

INTRODUCTION

Zearalenone [6-(10-hydroxy-6-oxo-trans—1-undecenyl)-|3-resorcylic acid
lactone] (Figure 1.1) is a mycotoxin produced by members of the genus Fusarium
after infection of corn and small grains (Mirocha et al., 1971; 1977; Hart et al.,
1982). When fed to animals, the compound causes hyperestrogenism with
symptoms such as enlargement of the uterus and nipples, vulvar swelling, vaginal
prolapse, and infertility (Mirocha and Christensen, 1974; Hidy et al., 1977). As a
consequence of these estrogenic effects, there is a need for routine screening of
agricultural commodities used for human and animal consumption.

Methods for the analysis of zearalenone in food and feeds include thin-layer
chromatography (TLC) (Gimeno, 1983; Scott et al., 1978; Swanson et al., 1984),
gas-liquid chromatography (Scott et al., 1978; Thouvenot and Morfin, 1979), high-
pressure liquid chromatography ( Scott et al., 1978; James et al., 1982; Turner et
al., 1983) and more recently immunoassays (T houvenot and Morfin, 1983; Liu et
al., 1985; Warner et al., 1986; Dixon et al., 1987; Teshima et al., 1990; Bennett et
al., 1994). Compared with other methods, immunoassays have several advantages
for rapid field tests including high specificity, sensitivity, facile sample preparation
and ease of use (Pestka et al., 1982). However, the development of antibodies
requires the use of animals, specialized cell culturing facilities, and an extensive
commitment of time and labor. Production of monoclonal antibodies from hybridoma
cell lines also requires specialized cell culture facilities and usually is time

consuming.

39

Advances in the field of recombinant antibody technology provide an
alternative means to engineer low cost antibodies with desirable affinity and
specificity by enabling one to manipulate the basic domain structure of the
immunoglobulin (lg) molecule. One of the most successful approaches is to display
scFv antibodies on filamentous phage (McCafferty et al., 1990; Barbas et al., 1991 ;
Clackson et al., 1991; Marks et al., 1991; 1992; Griffiths et al., 1993). ScFv is an
antigen-binding protein, composed of an immunoglobulin heavy chain variable
domain (VH) and a light chain variable domain (Vk or V,) joined together by a flexible
peptide linker (Figure 1.4). When expressed with phage protein g3p (fd 93 protein
of phage M13K07) as a fusion protein, a high affinity scFv producing phage clone
can be enriched by a procedure called "panning" (Marks et al., 1991 ; Hawkins et al.,
1992). In this part, we describe the molecular cloning, expression and
characterization of a functional scFv with high specificity and sensitivity to
zearalenone and its analogues, and the application of this scFv to the analysis of

zearalenone in spiked corn samples.

40

MATERIALS AND METHODS

Materials, strains and general methods

All chemicals and organic solvents were reagent grade or better.
Zearalenone was generously supplied by lntemational Minerals and Chemicals
Corp. (T erre Haute, IN). Zearalenone-ovalbumin and -keyhole limpet hemocyanin
(KLH) conjugates were prepared as described by Dixon et al. (1987) (Figure 2.1 ).
Anti-zearalenone hybridoma cell line 2GS-6E3-2EZ (Dixon et al., 1987) was kindly
supplied by M. A. Abouzied (Neogen Company, Lansing, MI). Plasmid
pCANTAB5E, E. coli TG1 and HB2151, M13K07 helper phage, horseradish
peroxidase conjugated sheep anti-M13 antibody and mouse anti-E tag antibody
were obtained from Phannacia Biotech (Piscataway, NJ). Cell culture components
and reagents, goat anti-mouse lgG peroxidase and rabbit anti-mouse lgG alkaline
phosphatase were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal
bovine serum, oligo(dT) cellulose columns and Not I restriction enzyme were
supplied by Gibco BRL (Gaithersburg, MD). PCR amplification primers were
synthesized by Gibco BRL(Gaithersburg, MD). The restriction enzyme Sfi I was
purchased from New England BioLabs (Beverly, MA). All DNA manipulations, if not

described, were carried out by standard procedures (Sambrook et al., 1989).

41

OH 0 Me
O
HO O

Carboxymethoxylamine
Zearalenone hemihydrochloride

Zearalenone-oxime 3
HO—N NHS
(RI-N=C=N—R1) 2—

EDC ’\

NOCHz| —O— N
OH ZRNHz (Protein)

140* I II

NOCHzZ—N—R + HO—N

Zearalenone-protein conjugte 2

 

 

 

Figure 2.1. Preparation of zearalenone immunogens through oxime chemistry
(based on Thouvenot and Morfin, 1983).

42

mRNA isolation

Hybridoma cell line 263-6E3-2EZ producing anti-zearalenone antibody
(Dixon et al., 1987) was cultured in macrophage conditioned DMEM medium
containing 20% (v/v) fetal bovine serum supplemented with 100 U/ml
penicillin/streptomycin in a 5% CO2 humidified chamber. After the cells were grown
to a density of 10° cells/ml, they were pelleted by centrifugation at 1000 x g for 5
min. For total RNA isolation, the pelleted cells were homogenized in RNA STAT-60
(TEL-TEST "B”, Inc., Friendswood, TX) at 5x10° cells/ml, followed by chloroform
extraction and isopropanol precipitation. mRNA was purified by affinity
chromatography with an oligo (dT)—cellulose column according to manufacturer

instructions.

cDNA synthesis, PCR amplification and scFv cloning

First strand cDNA was synthesized from mRNA template with M-Mulv
reverse transcriptase and random hexadeoxyribonucleotide [pd(N),,] primers
(Phannacia Biotech, Piscataway, NJ). The variable regions of heavy chain (VH) and
kappa light chain (VK) were amplified from first strand cDNA using Taq DNA
polymerase with 30 cycles of PCR (94°C, 1 min; 55°C, 1 min; 72°C, 2 min). The
primers used in the PCR amplification were based on Orlandi et al. (1989) and
Clackson et al. (1991), and modified for facilitating ligase-free UDG-ligation (Nisson
et al., 1991 ) (Table 2.1). VHBACK and du-VHFOR were used for amplification of V“;
dU-VKBACK and VKFOR were used for amplification of VK. A 93—bp DNA linker

(Phannacia Biotech, Piscataway, NJ), containing a sequence encoding a short

43

bL

re

f0

pr

flexible peptide, (Gly4Ser)3, was also amplified with LINKBACK and du-LINKFOR.
After gel purification with a QIAEX gel extraction kit (QIAGEN Inc., Chatsworth, CA),
25ng linker DNA and 75ng VK DNA were mixed and treated with 1 unit uracil DNA
glycosylase (UDG) (USB, Cleveland, OH) at 37°C in a 20ul reaction with 1x PCR
buffer for 1.5 hr. The UDG treated fragments were then scaled up to a 40pl PCR
reaction without primers and cycled 15 times (94°C, 1 min; 60°C, 1 min; 72°C, 2 min)
for joining the linker DNA with VK DNA. The joined linker-VK DNA was amplified in
a 100pl PCR reaction (94°C, 1 min; 60°C, 1 min; 72°C, 2 min) for 30 cycles with 75
pmol each VKFOR and du-LINKBACK. Gel-purified linker-VK DNA fragment (40 ng)
was joined with VH DNA (40 ng) into scFv (VH-Iinker-VK) DNA fragment in a 20 pl
PCR fill-in reaction after UDG treatment. The assembled scFv DNA was amplified
with 75 pmol each VHBACK and VKFOR in a 100 pl PCR amplification reaction for
30 cycles as described above. Finally, the assembled scFv products were gel-
purified and reamplified with restriction site tagged primers (RS Primers Mix)
(Phannacia Biotech, Piscataway, NJ) to append an Sfil site on the 5' end and a Notl
site on the 3' end of the scFv DNA. The scFv DNA products were digested with Sfil
and Notl restriction enzymes, gel purified and then ligated into the phagemid
pCANTAB5E (digested with these same enzymes).

44

Table 2.1: Sequences of oligonucleotide PCR primers for
PCR amplification of V", VK, and linker DNA*

 

VHBACK: 5' AGGTSMARCTGCAGSAGTCWGG-3'

dU-VHFOR: 5' UGAGGAUACGGUGACCGUAGUACCUUGGCCCC-3'
dU-VKBACK: 5' GACAUCGAGCUCACUCAGUCUCCA—S'

VKFOR: 5' CCG'ITI'BAKYTCCARC'ITKGTSCC—3'

LINKBACK: 5' GGTACTACGGTCACCGTATCCTCAGGTG-3'

dU-LINKBACK: 5' GGUACUACGGUCACCGUAUCCUCAGGUG-3'
dU-LINKFOR: 5' AGACUGAGUGAGCUCGAUGUCCGAUCC-3'

 

*: U=deoxyuracil; R=A or G; Y=C or T; M=A or C; K=T or G; S=C or G; W=A or T;
B=T or C or G;

45

ScFv antibody expression and panning-elution selection

To display scFv as a fusion protein with E-tag and M13 g3p (there is an
amber stop codon between E-tag and fd 93 in pCANTAB5E), the ligated products
were used to transform E. coli TG1 competent cells which is an amber suppressor
strain (supE). Transformed cells were plated on SOB medium (Sambrook et al.,
1989) containing 100 pg/ml ampicillin and 2% glucose and incubated at 30°C
overnight. Colonies were pooled and infected with M13K07 helper phage in 2x YT
medium (Sambrook et al., 1989) containing 100 pg/ml ampicillin and 50 pg/ml
kanamycin to rescue the phagemid with its scFv gene inserts and to display scFv
fusion protein on the surface of the recombinant phages. The recombinant phages
from the supernatant were filtered through a 0.45 pm-pore-size filter.

For panning-elution selection (Figure 2.2), wells of sterile polystyrene
lmmulon-4 microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated
overnight (4°C) with 100 pl of zearalenone-ovalbumin conjugate (10 pg/ml) in 0.05
M carbonate-bicarbonate buffer (pH 9.6). Plates were washed four times by filling

each well with 300 pl of 0.05% (v/v) Tween-20 in 0.01 M PBS (PBS-T) and

aspirating the contents. Non-specific binding was blocked by addition of 300 pl
“blocking buffer" [1% ovalbumin (w/v) dissolved in PBS (PBS-OVA)] to each well
for 1 hr at 37 °C and then washing 6 times with PBS-T. Recombinant phages
(about 5 x 1010 pfulml) were then added to the walls (100 pllwell) of a zearalenone-
ovalbumin coated plate. The plates were incubated for 2 hr at 37°C followed by ten

washes with PBS and ten washes with PBS-T. One hundred microliters of free

46

Fic

 

 

 

 

 

lie-Infection and re-empllficetlon

 

Figure 2.2. Panning-elution selection procedure for anti-zearalenone scFv phage
in zearalenone-ovalbumin (ZEN-OVA) coated lmmunlon-4 microtiter
wells.

47

268
OOH

vvnl

syn
vver
affii

68C

liEi
FHBJ
gfll
fliej
adc
0(3c
set;
thrc

detr

(3or

and

"Her

zearalenone (0, 0.01, 0.1, 1.0, 5.0, 10.0, 15.0, and 50.0 pg/ml) diluted with PBS
containing 1.5% methanol were added to the wells and incubated at 37 °C for 1 hr
with rotary shaking (150 rpm) to elute the bound phages. The zearalenone eluted
phages were collected and passed through a 0.45 pm-pore-size Iow-protein-binding
syringe filter (Gelman Sciences, Ann Arbor, MI). These zearalenone specific phages
were used to reinfect E. coli TG1 cells for subsequent rounds of selection. The
affinities of recombinant phage antibodies from pooled or individual colonies after
each round of selection was tested with a modified Cl-ELISA described below.
Recombinant phages from selected clones were used to infect E. coli
HBZ151 for production of soluble scFv antibodies. Briefly, the infected E. coli
HB2151 cells were cultured in SB medium (35 g/l tryptone, 20 g/l yeast extract, 5

g/l NaCl) supplemented with 100 pglml ampicillin at 30°C with 250 rpm shaking until

they reached an A600 of 0.5. lsopropyl-B-D-thiogalactopyranoside (IPTG) was then
added to 1 mM final concentration and the cells were incubated on a shaker at 30
°C overnight. Supematants containing the extracellular soluble scFv antibodies were
separated from the cell pellets by centrifugation at 1500 x g for 15 min, and filtered
through a 0.45 pm-pore-size filter. The affinity of soluble scFv antibodies was
detected with a modified Cl-ELISA described below.

Competitive Indirect ELISA (Cl-ELISA)
Several Cl-ELISAs were developed and used to determine the sensitivities
and specificities of the scFv antibodies (phage-associated or soluble forms). Briefly,

microtiter plates were coated and blocked as described above. Next, 50 pl of

48

zearalenone standard (or analogues) at various concentrations dissolved in PBS
containing 1% methanol was added to the coated wells and simultaneously
incubated with 50 pl of phage-associated (about 5 x 1010 pfulml in 2x YT medium)
or soluble scFv antibody (various dilutions with PBS-OVA) or parent monoclonal
antibody (diluted 1:5 with PBS-OVA) from the supernatant of hybridoma cell culture
for comparison. The plates were incubated for 1 hr at 37 °C then washed 6 times
with PBS-T. The amount of bound phage-associated scFv antibody was determined
by the addition of 100 pl anti-M13-HRP conjugate diluted 1:2500 with PBS-OVA.
The amount of soluble scFv antibody bound was determined by incubation with 100
pl mouse anti-E tag (diluted 122500 with PBS-OVA) at 37°C for 1 hr followed by
washing (six times) and a subsequent incubation with goat anti-mouse lgG HRP
conjugate (diluted 1:500 with PBS-OVA). The bound monoclonal antibody was also
detected by the addition of 100 pl goat anti-mouse lgG HRP conjugate (diluted
1:500 with PBS-OVA). The plates were incubated for 1 hr at 37°C and then washed
6 times with PBS-T. The amounts of anti-M13-HRP and goat anti-mouse IgG HRP
conjugates bound were assessed by the addition of 100 pl of 2,2'-azinobis(3-
ethylbenzthiazoline sulfonic acid) (ABTS) substrate (Pestka et al., 1982). The plates
were incubated for 15 min for color development and the absorbances were

determined at 405 nm with a microtiter plate reader (Molecular Devices Corporation,

Menlo Park, CA).

49

ar

fol

ins
tOIl

Vis

SDS-PAGE and Western blot analysis

Supematants, containing the extracellular soluble scFv antibodies in SB
medium from E. coli HB2151 cell culture, were precipitated with trichloroacetic acid
(T CA) at a final concentration of 10% (w/v) on ice for 20 min. After centrifugation in
a microcentrifuge at 14,000 rpm for 10 min, the pellet was resuspended in 15 pl of
0.5 M Tris buffer (pH 8.0), heated for 5 min at 95°C after adding 5 pl loading buffer
(4 x), and subjected to SDS-12% polyacrylamide gel electrophoresis with a 0.5 mm
thick gel in a Bio-Rad Mini-Protein l| Eletrophoresis System (Bio-Rad Laboratories,
Hercules, CA). Prestained low range SDS-PAGE standards (Bio-Rad laboratories)
were used to calibrate protein mobilities. Two identical gels were run in parallel.
After separation, the protein bands in one gel were stained with Coomassie brilliant
blue G-250, those in the other gel were transferred to a nitrocellulose membrane
(Schleicher & Schuell, Kneene, NH) in a Multiphor II Electrophoresis System (LKB
Produkter AB, Bromma, Sweden). The transblotted membrane was probed with
anti-E tag antibody (about 8 pg/ml in PBS-OVA containing 0.05% Tween 20)
followed by incubation with rabbit anti-mouse lgG alkaline phosphatase conjugate
(Sigma Chemical Company, St. Louis, MO) as described by manufacturer’s
instructions. Nitroblue tetrazolium and 5-bromo-4-chloro-3- indolyphosphate-p-
toluidine (NBT/BCIP)(Pierce, Rockford, IL) were used as the substrate for

visualization.

5O

Sequence analysis

Plasmid DNA of anti-zearalenone producing clones was isolated from E. coli
HBZ151 by alkaline lysis. VH and VK DNA portions in the plasmid were sequenced
on both strands with the pCANTAB5 sequence primer set (Phannacia Biotech,
Piscataway, NJ) by using Taq cycle sequencing and dye terminator chemistry at the
MSU Sequencing Facility. Sequence comparisons were performed by searching the

Kabat Database at the Internet Web site (http:/limmuno.bme.nwu.edu).

Cl-ELISA for spiked corn extracts

The procedures for extraction of corn and spiking were similar to those
described by Liu et al. (1985). Briefly, zearalenone-free homogeneous ground corn
was extracted with five volumes (w/v) of methanol-water (70:30) for 5 min, with
constant stirring. The mixture was immediately filtered through a Whatman No. 4
filter paper and the filtrate was spiked directly with zearalenone, or the filtrate was
diluted 2 times with 1x PBS and then spiked with zearalenone. The spiked extracts
were then subjected to Cl-ELISA using the soluble scFv antibodies and monoclonal
antibody in a similar procedure as described above with the following modifications.
(i) Since anti-E-tag antibody, which was used as a second antibody to detect the
bound scFv antibodies, exhibited nonspecific binding to methanol treated carrier
protein ovalbumin, zearalenone-KLH conjugate was used in the Cl-ELISA for
methanol extracted corn samples. (ii) Coated wells were blocked by PBS

containing 10% nonfat dry milk. (iii) Antibodies were diluted in PBS containing 10%

51

nor

pFC

Nu

Ul’l

nonfat dry milk. The spiking experiment was repeated once with the same

procedure.

Nucleotide sequence accession numbers

The sequences determined for VH and VK from this clone are available

under GenBank database accession numbers U74671 and U74672, respectively.

52

RESULTS

PCR amplification

VH and VK cDNA from hybridoma cell line 2GS-6E3—2EZ were amplified by
PCR and assembled into an scFv encoding DNA fragment (Figure 2.3).
Amplification of VK generated a major DNA fragment with the expected length
(about 324 bp), while VH generated an expected 340 bp fragment and two other
closely migrating fragments. The PCR assembly of linker-Vk produced two major
closely migrating fragments (lane 5 in Figure 2.3) with approximately the expected
size plus a minor band much larger than the expected product. The two major
linker-Vk fragments, which might be generated by alternative priming of the linker
DNA with its nearly identical repeats, were collectively isolated, purified, and then
PCR assembled with VH products (the mixture of three major bands in lane 2 of
Figure 2.3) to form the scFv fragments in lane 6 of Figure 2.3. For the assembly of
scFv, a preliminary experiment with a one step PCR fill-in reaction failed to join VH,
linker, and VK . The modified two-step procedure described in Materials and

Methods greatly improved the joining.

53

Figul

1234567

scFv
"" 750bp

 

Figure 2.3. VH and VK amplification and assembly of scFv. Lanes are: (1): VH
marker (pUC18NH, EcoR l/Xba | digested, Pharmarcia Biotech);
(2): VH PCR product; (3): VK PCR product; (4): Linker PCR product;
(5): Linker-VK PCR product; (6): VH-Linker-VK (scFv) PCR product;
(7): scFv marker (pUC18/A1OB, Sfi l/ Not I digested, Pharrnacia
Biotech).

Phag

town
wdht
used
stop I
fluap
flfllE
aHou
wens
eune
phag

after

PUC

FigL

Phage display of scFv antibody and panning-elution selection

Two major scFv DNA fragments in lane 6 of Figure 2.3 were collectively
isolated, purified, digested, then ligated into pCANTABSE, and translationally fused
with the E-tag DNA fragment (Figure 2.4). The resulting library, named pQY1, was
used to transform E. coli TG1 cells (amber suppression strain), in which the amber
stop codon between the E-tag DNA sequence and fd 93 was read through, allowing
the production of scFv E-tag-g3p fusion protein. In the presence of helper phage
(M13K07), scFv fusion products were displayed on the recombinant phage tips,
allowing for affinity selection with panning-elation. In lmmulon-4 microtiter plate
wells coated with zearalenone-ovalbumin, the bound recombinant phages were
eluted by free zearalenone at a minimum concentration of 1 pg/ml. The eluted
phages were used for elution in the sequential round of panning-elution selection

after re-amplification.

TAG (amber stop codon)

 

 

133

Figure 2.4. pQY1 construct. PCR amplified scFv was cloned into Sfil and Nail
sites of pCANTAB5E, a pUC119 based vector from Phannacia
Biotech. The linker DNA sequence was
5'GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTG
GCGGATCG-3', which encoded a short peptide [(Gly4Ser)3].

55

low :
a ole
(Figl
bind
rour
rour
eac
isol:
am;
iron
dist
binr

fror

Sol

bet
SOIL
r90
bInt
Of 5

IPT

Prior to affinity selection, the pooled recombinant phage library exhibited very
low zearalenone-binding activity (Figure 2.5). After one round of affinity selection,
a clear enrichment for scFv clones expressing zearalenone-binding was observed
(Figure 2.5). The lC50 (concentrations of free zearalenone required for inhibition of
binding by 50%) for preselection phage, first round selected phage and second
round selected phage were >1000, 31, and 28 ng/ml, respectively. The second-
round selection mainly increased yield of functional scFv fusion protein. Following
each round of panning-elution selection, individual clones (in TG1) were randomly
isolated and the scFv DNA insert in these clones was confirmed by PCR
amplification and restriction digestion with Sfil and Notl. Three individual clones
from each selection group were rescued with helper phage M13K07 for scFv phage
display. When the resultant recombinant phages were tested for zearalenone
binding in CI-ELISA, all three preselection clones were negative whereas those

from first and second round selection were positive.

Soluble scFv antibody production and characterization

In the nonsuppressor strain, E. coli HB2151(supE'), the amber stop codon
between E- tag and fd 93 in scFv clones is recognized as a stop codon and a
soluble scFv-E tag fusion protein is produced as a consequence. Three individual
recombinant phage clones (the same clones tested above for zearalenone
binding) from each selection group were used to infect HB2151. The expression
of scFv E-tag fusion protein in the resultant HB2151 clones was induced by

IPTG, and soluble scFv antibodies were secreted to the culture supematants.

56

Fig

 

 

-O—pre-selection
—A— lst round selection

0.8 ~ + 2nd round selection

 

 

 

0.6 -

A405

0.4 i

0.2 ..

 

 

1 10 100 1000 10000
Zearalenone (ng/ml)

Figure 2.5. Zearalenone-binding activities of recombinant phage following each
round of panning-elution selection measured by CI-ELISA in
zearalenone-ovalbumin coated lmmulon—4 microtiter wells. Bound
phages were detected by horseradish peroxidase conjugated sheep
anti-M13 antibody.

57

A405

0

Fig

 

  
 
  
 

Figure 2.6.

120

 

-<>-(IY1.I

‘x‘QYl-Z 100 - +QY1.12
+QY1.12

-O— .
-O—QY1.5 QY1 5

-HD-nukb

    

80-

  

60-

Percent of inhibition

 

 

 

 

l 10 100 1000 I 10 100 1000

Zearalenone (ng/ml) Zearalenone (ng/ml)

Sensitivity of soluble scFv antibodies tested in Cl-ELISA in
zearalenone-ovalbumin coated Immulon-4 microtiterwells. QY1 .1 and
QY1.2 ( no dilution) were from preselection; QY1.5 (5x dilution) was
from first round of panning-elution selection; QY1.12 (10 x dilution)
was from second round of panning-elution selection; mAb
(supernatant of hybridoma cell culture) was diluted 5 times. Bound
soluble scFv antibodies were detected by mouse anti-E tag antibody,
followed by horseradish peroxidase conjugated goat anti-mouse lgG.
A: Absorbance reading at 405 nm. B: Percent of binding inhibition
determined by dividing sample absorbance by control absorbance
x 100.

58

Table 2.2: Comparison of cross-reactivities between scFv antibody and
mAb toward zearalenone analogues

 

 

 

 

mAb scFv QY1.5

Analogue IC50 Cross-reactivity* IC50 Cross-reactivity
(no/ml) (%) (no/ml) (%)
Zearalenone 17 100 14 100
a-zearalenol 66 26 17 82
B-zearalenol 159 1 1 54 26
a-zearalanol 212 8 23 62
B—zearalanol 175 10 54 26

 

*: Cross-reactivity defined as (lemmbm)/( leo mm”) x 100 %.

59

In

In

The zearalenone-binding pattern of soluble scFv antibodies from these nine
HB2151 clones was the same as their parent phage scFv antibodies. Cl-ELISA
results for four typical scFv clones are shown in Figure 2.6A. Two of the scFv
antibodies (QY1.5 and QY1.12 from first round and second round selection,
respectively) exhibited zearalenone-binding affinities similar to those of the parent
monoclonal antibody (Figure 2.6B). leo for scFv QY1.5, QY1.12 and their parent
monoclonal antibody were 14, 35, 17 ng/ml, respectively. Compared with the parent
monoclonal antibody, however, soluble scFv QY1.5 exhibited similar sensitivity but

higher relative cross-reactivities to hydroxylated zearalenone analogues (Table 2.2).

SDS-PAGE and Western blot analyses

An approximately 32 KDa scFv-E tag fusion protein was expressed from
HB2151/pQY1.2, HB2151/pQY1.12 and HB2151/pQY1.5, while HB2151/pQY1.1
expressed a smaller protein, which probably lacked an E tag and could not be
detected by anti-E tag antibody (Figure 2.7). Among the two positive zearalenone
binding clones, HB2151/pQY1.12 expressed higher yields of scFv than
HB2151/pQY1.5; however, the affinity of its scFv antibody (QY1.12) for

zearalenone appeared slightly lower than that of QY1.5 (Figure 2.5B).

Sequence analysis
Clone pQY1.5, producing scFv with the highest affinity for zearalenone in
HB2151, was chosen for DNA sequencing. The deduced amino acid sequences of

VH and VK in pQY1.5 are shown in Figure 2.8. According to the amino acid

60

 

Figure 2.7.

SDS-PAGE gel and Western blot analysis of scFv expression.
(A): Supematants of QY1.1 (lane 1; 0.3 ml), QY1.2 (lane 2; 0.3 ml),
QY1.12 (Iane3; 0.3 ml) and QY1.5 (lane 4; 0.6 ml) concentrated in
10% trichloroacetic acid were subjected to SDS-PAGE and stained

with Coomassie brilliant blue G-250. (B): Western blot of duplicate
SDS/PAGE gel.

61

sequences, the VH was classified as mouse heavy chain subgroup lll (D), while VK
fell into mouse kappa light chain subgroup V (12) (Kabat et al., 1991). The
sequences of VH and VK were screened against the entries of lg heavy chain and
lg kappa light chain respectively in the Kabat Database. The VK sequence was
closely related to that of a genomic DNA clone VK23.32, a member with no identified
binding specificity in gerrnline gene VK23 family (Lawler et al., 1989). There was
97% nucleotide sequence identity between VK of pQY1.5 and VK23.32 with only 9
nucleotide differences, resulting in 4 amino acid changes. Among the 9 changed
nucleotides, 4 were in the framework region (FW) 1, and were attributed to the use
of a degenerate VKBACK primer, 3 were in FW3, and a single silent nucleotide
change (causing no amino acid sequence change) occurred in both complementary-
detennining regions (CDR) 2 and 3. This implied that V, 23.32 might be the germ
line gene for Vk of scFv QY1.5. Thus, few somatic mutations in the kappa light
chain occurred during the in vivo affinity maturation of its parent anti-zearalenone
antibody. However, the sequences of CDRs (especially CDR2 and CDR3) in the VH
of pQY1.5 were not related to any known members in the database. The closest
VH sequence was that of L3 10A (Kettleborough et al., 1994), an antibody with
specificity to human epidermal growth factor receptor. But there was only 68% and
47% sequence identity between scFv OY1.5 and L3 10A in their VH CDRs at the
nucleotide and amino acid levels, respectively, although they had 96% identity in

the FWs at both nucleotide and amino acid levels.

62

 

DlELTc

GIPSR]

Figul

A. VH sequence:

FWl CDRI FW2 CDR2
MA QVKLQESGGGLVQPGGSLKLSCAASGFTF S NYGMS WVRQTPDKRLEFVA NINGNGGKTYYPGSVKG

 

FW3 CDR3 FW4

RFTISRDNAKNTLYLQMSSLKSEDTAMYYCVR VAFDGYYDDF WGQG'ITVTVSS

 

B. VK sequence:

FWl CDR] FWZ CDR2
DIELTQSPATLSVTPGDRVSLSC RASQSISDYLH WYQQKSHESPRLLIK YASQSIS

FW3 CDR3 FW4

GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC QNGHSFPPT FGGGTKLEIK

Figure 2.8. Deduced amino acid sequences of VH and VK in scFv QY1.5.

Complementary-determining regions (CDR) and framework regions
(FW) are indicated.

63

AP

QY
mc
EL

28

to

26

3F

Application of scFv QY1.5 to a spiked corn extract

Using zearalenone-KLH conjugate coated microtiter plates, soluble scFv
QY1.5 showed higher sensitivity to zearalenone in corn extracts than its parent
monoclonal antibody when the final methanol concentration was 17.5% in the Cl-
ELISA solution (Figure 2.9). Corn extracts containing as little as 10 ng
zearalenone/ml caused a visually distinct inhibition of absorbance with soluble scFv
OY1.5 in Cl-ELISA. However, for undiluted corn extract (35% methanol in the final
CI-ELISA solution), soluble scFv QY1 .5 failed to detect any level of zearalenone in
Cl-ELISA because zearalenone specific binding of QY1.5 was eliminated by
methanol at concentrations higher than 25% in the final Cl-ELISA solution (Figure
2.10). In contrast, the parent monoclonal antibody exhibited much highertolerance
to methanol destabilization (Figure 2.10). Regardless, scFv QY1.5 did bind
zearalenone with high specificity and sensitivity at practical levels of methanol
concentration (less than 20%). Thus, the scFv antibody described here should be
applicable for routine screening of zearalenone in corn and perhaps other cereal

grains.

100

Percent of inhibition

Figure 2.9.

80‘

60~

4o~

20-

 

 

 

 

 

 

 

 

 

0.1 l 10 100 1000 10000

Zearalenone (ng/ml)

Zearalenone sensitivities in spiked corn extracts with monclonal
antibody and soluble scFv antibody OY1.5 measured by Cl-ELISA in
zearalenone-keyhole limpet hemocyanin conjugate coated Immulon-4
microtiter wells. Both scFv QY1.5 and mAb were diluted 1:4 in PBS
containing 10% nonfat dry milk. Zearalenone in ground corn was
extracted with 5 volumes (wt/vol) of methanol-water (70:30) and the
extracts were then diluted 1:1 with PBS.

65

 

 

A405

 

 

 

 

 

 

0 I I I I I I
0 5 10 15 20 25 3O 35

 

Methanol concentration in test well, %

Figure 2.10. Effects of methanol on the binding of mAb and scFv QY1.5 to
zearalenone-keyhole limpet hemocyanin conjugate. lmmulon-4
microtiter wells were coated with zearalenone-keyhole limpet
hemocyanin conjugate. Bound soluble scFv antibody was detected by
mouse anti-E tag antibody, followed by horseradish peroxidase
conjugated goat anti-mouse lgG. Bound monoclonal antibody was
detected by horseradish peroxidase conjugated goat anti-mouse I96.

66

from
and

succ
and '

anut

anflt
fort
proc
i~hk
very
dev.

Dior

mR
"Tel
96o
98v
an”

all

DISCUSSION

This study describes the successful isolation and cloning of VH and VK genes
from a zearalenone-specific hybridoma, their soluble expression as an scFv protein,
and their use in a zearalenone Cl-ELISA. To our knowledge, this is the first
successful example of the development of a recombinant antibody for mycotoxins,
and we think it could serve as a model for development and construction of novel
antibodies for mycotoxins and other potentially toxic residues.

When a hybridoma is used as a mRNA source for construction of scFv
antibody, it is most desirable to use a cell line that produces high-affinity antibody
for the target hapten or antigen. The hybridoma cell line we used in this study
produced monoclonal antibody with high affinity (le0=17 ng/ml) for zearalenone,
which made the cloning of high affinity scFv antibody possible. In contrast, only a
very low sensitive scFv antibody specific for the mycotoxin fumonisin B, was
developed in a previous study because, in part, the hybridoma cell line used
produced a lower affinity antibody specific to fumonisin B, (Zhou et al., 1996).

Although hybridomas producing high affinity antibodies are good sources of
mRNA for the facile cloning of desired variable region (V region) genes, additional
irrelevant transcripts are possible. Thus, it was notable that amplification of the VH
gene from hybridoma 2G3-6E3-2EZ, which produces anti-zearalenone antibody,
gave three different PCR products. One of these products may have been the
amplified product from the fusion partner, myeloma cell line NS-1, which produces

an aberrant V,, transcript with a 50 nucleotide deletion at FW3-CDR3 boundary

67

(Thammana, 1994). Other irrelevant V regions might possibly result from non-
productive rearrangements. Practically, a hybridoma may contain several different
cell populations as a result of recombination during extended culture after limiting
dilution. So V regions which are productive but do not recognize the antigen of
interest may also be isolated. Additionally, modifications could be generated in the
PCR amplification and assembly because of the use of degenerate primers and Taq
DNA polymerase with low fidelity. Affinity selection was therefore necessary for
isolation of the variable regions with the desired specificity. Like the scFv antibodies
before panning elution selection in this study, the fumonisin B, specific recombinant
scFv antibody (Zhou et al., 1996), selected only by panning without affinity elution
selection (through hapten elution), were 10 to 100 times less sensitive compared
with anti-fumonisin B, polyclonal or monoclonal antibodies. Clackson et al. (1991)
also showed that affinity elution with free antigen or hapten elution after panning
greatly enriched clones producing high affinity scFv antibody.

Since only a few species of mRNA transcripts from a specific hybridoma cell
line contain V regions, a few rounds of panning-elution selection should select the
desired V regions. In this study, a functional anti-zearalenone scFv protein with
high affinity was isolated after only one round of panning-elution selection.
Sequential selections mainly appeared to enrich clones with a higher yield of scFv
protein, not necessarily improving the affinity.

Theoretically, a scFv will have similar specificity and sensitivity as its parent
immunoglobulin molecule. In this study, soluble scFv antibody QY1.5 exhibited an

affinity to zearalenone similar to that of its parent monoclonal antibody, but it was

68

showr
these
at al..
partic
gene
and
SBQI
reve

seqr

COV

to t
of s
effi
of

de

ml

at

shown to have higher cross-reactivity to hydroxylated zearalenone analogues. Since
these analogues are naturally occurring toxic metabolites of zearalenone (Mirocha
et al., 1981; Hagler et al., 1979), the ability of the scFv antibody to detect them is
particularly advantageous when detection of these analogues is desirable. In
general, a change in specificity and sensitivity could result from sequence changes
and conformation differences between scFv and the monoclonal antibody.
Sequence changes may occur during the scFv construction procedures, including
reverse transcription, PCR amplification, enzyme digestion and ligation. Even if the
sequences of V,, and VK in the scFv are the same as those naturally occuring in an
lgG molecule, their conformations may also differ because scFv has V,, and VK
covalently coupled by a flexible peptide linker while natural lgG molecule does not.

ScFv QY1 .5 also differed from its parent monoclonal antibody in its sensitivity
to the destabilizing influence of methanol, which drastically decreased the binding
of scFv QY1.5 to zearalenone. Muller et al. (1994) observed a similar destabilizing
effect of glycerol on scFv, which promoted the dissociation of two variable domains
of scFv protein but did not affect the domain structure of scFv. In contrast,
destabilization was not observed for Fab fragments and whole monoclonal antibody
molecules in that study. It was believed that the constant domains in Fab or
monoclonal antibody may provide additional stabilization of the molecular structure
at the antigen binding site. Some general strategies for protein stabilization may
be used to stabilize scFv protein by strengthening the interactions between VH and

VK domains. These include engineering of stronger hydrogen bonds, disulfide

69

bonds, metal binding sites, and increasing the hydrophobic area or electrostatic
interactions (Raag and Whitlow, 1995).

It was notable that the VH sequence of scFv QY1.5 exhibited numerous
sequence differences from sequenced members in the antibody database. As might
be expected, the changes are clustered in the CDRs, especially in CDR2 and
CDR3. This implies that CDR2 and CDR3 in the V,, of scFv QY1.5 may play an
important role in determining the specificity for zearalenone binding. In contrast, the
VK in scFv QY1.5 may contribute less to the specificity, since there were few
differences in its sequence compared with its possible germ line gene. The latter
may have a broad range of specificities without somatic mutations.

As immunoassays become a more common method forthe routine screening
of mycotoxins and other natural toxicants in food and agriculture products, larger
supplies of low-cost high affinity antibodies will be needed. The recombinant
antibody technique provides a promising alternative for producing low-cost
antibodies with the desirable specificity and sensitivity. The recombinant antibody
scFv QY1.5 has high sensitivity and specificity to zearalenone and its analogues,

and should be readily applicable to the routine screening for these mycotoxins.

70

PART III

ATTEMPTS TO CLONE SINGLE-CHAIN FV (SCFV) ANTIBODIES AGAINST
FUMONISIN B, FROM IMMUNIZED MICE

71

ABSTRACT

ATTEMPTS TO CLONE SINGLE-CHAIN FV (SCFV) ANTIBODIES
AGAINST FUMONISIN B, FROM IMMUNIZED MICE

High affinity (le0, 0.27 pg fumonisin B, lml) mouse polyclonal antibodies
were readily generated after immunization with fumonisin B, -cholera toxin (FB,-CT)
conjugate but not produced with fumonisin B,-keyhole limpet hemocyanin (FB,-KLH)
conjugate immunization. From the splenic cDNA of FB,-CT immunized mice,
variable region genes of heavy- (VH) and kappa light-chain (VK) were successfully
amplified by PCR with previously designed primers (Yuan et al., 1997). The V,, and
VK genes were assembled with linker DNA to form scFv DNA fragments by PCR
and cloned in a phage-display vector. However, panning-elution selection from this
scFv phage-display library did not result in enrichment of PB, specific phage clones.
Different FB, conjugates, phage incubation times and F3, concentrations in the
eluting solution were investigated for their effects on specific binding and elution.
No conditions were found suitable for FB, specific scFv phage selection in the
panning-elution selection procedure. The soluble form of scFv proteins from
approximately 300 randomly selected individual clones did not show any FB,
specific binding activity either. An alternative method is suggested to clone

functional FB, specific scFv antibodies.

72

INTRODUCTION

Fumonisins are toxic secondary metabolites produced by certain species of
Fusarium during pathogenesis on corn and sorghum (Sydenham et al., 1990).
Interest on fumonisins has intensified recently because of its cytotoxic properties
and carcinogenic potential (T hiel et al., 1991; Gelderblom et al., 1993). Six different
fumonisins, fumonisin A,, A,, B,, B,,, B,,, and B,, have been chemically characterized
(Figure 1.2) (Bezuidenhout et al., 1988; Cawood et al., 1991; Plattner et al., 1992).
Fumonisin B, (FB,) is the most toxic and primary fumonisin produced by Fusarium
moniliforme (Gelderblom et al., 1988b). FB, produces characteristic effects in
different animal species, including hepatic cancer in rats, brain lesions in horses
(equine leukoencephalomalacia, ELEM), and pulmonary lesions in pigs (porcine
pulmonary edema, PPE).

Elimination of fumonisins from human and animal food requires detection
and diversion of contaminated raw materials from feed and finished food use.
Commonly used chemical analytical methods for detecting fumonisins are thin layer
chromatography (TLC), high performance liquid chromatography (HPLC), and mass
spectroscopy (MS). Fumonisins do not absorb ultraviolet light and do not fluoresce;
therefore, derivatization reactions are required before detecting the toxins with
chromatographic techniques (Geldeblom et al., 1988a; Shephard at al., 1990;
Cawood et al., 1991; Rottinghaus et al., 1992; Scott and Lawrence, 1992).
Regardless of sensitivity and selectivity, these chemical analytical methods are

generally expensive, complex, and time consuming (Pestka et al., 1995). More

73

recently, immunoassays have proven to be useful in screening for mycotoxins.
Compared to the chemical analytical methods, immunoassays have several
advantages for rapid field tests, including high specificity, sensitivity, facile
sample preparation, and ease of use (Pestka et al., 1995). As a consequence, both
polyclonal (Azcona-Olivera et al., 1992a) and monoclonal (Azcona-Olivera et al.,
1992b) antibodies to FB, have been developed and applied to the enzyme-linked
immu nosorbent assays (ELISAs) (Elissalde et al., 1995; F ukuda et al., 1994; Pestka
et al., 1994a; Tejada-Simon et al., 1995).

Such developments have lead to a great demand for antibodies in the
fumonisin immunoassays. However, the limit of detection with most current
antibodies is not sufficiently adequate for screening fumonisins at <1 ppm in
conventional ELISA or multianalyte assays. Furthermore, the development of these
natural antibodies requires the use of animals, specialized cell culturing facilities,
and an extensive commitment of time and labor.

It is now theoretically feasible to engineer low cost antibodies with desirable
affinity and specificity characteristics by using recombinant DNA technology to
manipulate the basic domain structure of immunoglobulin molecule (Hoogenboom
et al., 1992; Lerner et al., 1992). Using filamentous phage to display antibodies,
antibody fragments can now be engineered from antibody V-gene repertoires
(McCafferty et al., 1990; Clackson et al., 1991; Marks et al., 1991) and high affinity
clones can be selected by a procedure called “panning” (Marks et al., 1991;

Hawkins et al., 1992).

74

Previously, several single-chain Fv clones were developed in our laboratory
using mRNA from either spleen cells of mice immunized with FB,-bovine serum
albumin (FB,-BSA) conjugate or from an existing hybridoma cell line that produces
anti-FB, antibody (Zhou et al., 1996). However, these scFv antibodies had very low
affinities to fumonisins and were even 10 to 100 times less sensitive than the
parent natural antibodies. The failure to develop high affinity scFv antibodies to
fumonisins in the previous study might be due to (i) the use of a hybridoma cell line
or immunized mice which produced low affinity antibodies; (ii) only panning was
used in the selection, without affinity elution. Based on the successful development
of anti-zearalenone scFv antibodies (Yuan et al., 1997), it is desirable to use a cell
line or immunized mice that produce high-affinity antibodies to start scFv fragments
cloning. For the selection of specific scFv antibodies, it is also critical to conduct an
affinity elution by free hapten after the regular panning. In this study, the possibility
of developing high affinity scFv antibodies to fumonisin B, was investigated by using
(i) spleen mRNA from immunized mice that produce high affinity antibodies to

fumonisin 3,; (ii) affinity elution with free fumonisin B, after the regular panning.

75

MATERIALS AND METHODS

General procedures

All chemicals and organic solvents were reagent grade or better. Fumonisin
B,, cholera toxin (CT), ovalbumin (OVA), bovine albumin (BSA), glutaraldehyde,
goat anti-mouse lgG peroxidase and rabbit anti-mouse lgG alkaline phosphatase
were purchased from Sigma Chemical Co. (St. Louis, MO). lnject" keyhole limpet
hemocyanin (KLH) was purchased from Pierce Chemical Company (Rockford, IL).
Plasmid pCANTAB5E, E. coli TG1 and HB2151, M13K07 helper phage,
horseradish peroxidase conjugated sheep anti-M13 antibody and mouse anti-E tag
antibody were obtained from Pharmacia Biotech (Piscataway, NJ). Oligo(dT)
cellulose columns and Not I restriction enzyme were supplied by Gibco BRL
(Gaithersburg, MD). PCR amplification primers were synthesized by Gibco
BRL(Gaithersburg, MD). Restriction enzyme Sfil was purchased from New England
BioLabs (Beverly, MA). All DNA manipulations, if not described, were carried out by

standard procedures (Sambrook et al., 1989).

Preparation of Immunogens

Fumonisin B, was conjugated to KLH (FB,-KLH), cholera toxin (FB,-CT),
ovalbumin (FB,-OVA), and bovine semm albumin (FB,-BSA) with glutaraldehyde
linkage via a free amino group (Figure 3.1) as described by Azcona-Olivera et al.
(1992a). FB,-KLH and FB,-CT conjugates were used as immunogens, and FB,-

OVA and FB,-BSA conjugates were used as solid-phase antigens for affinity

76

selection. Briefly, the coupling reaction was carried out at 4°C in 0.01 M phosphate-
buffered saline (pH7.4) (PBS). F3, was added to a 1-mglml suspension of protein
at a molar ratio of 50:1 (toxin/protein), and then an equal volume of glutaraldehyde
(2% vol/vol) was added dropwise with constant stirring. After 1 hr, the reaction was
stopped and the bond stabilized by reduction via the addition of sodium borohyd ride
to a final concentration of 10 mg/ml. One hour later, the mixture was dialyzed for 72
hr (three changes) against 0.01 M PBS (pH7.4). The conjugates then were

aliquoted in fractions of 1 mg (total protein) and stored at -20°C until required.

Animal Immunization

Female BALB/c mice (7 to 8 weeks of age) were immunized by
intraperitoneal injections. Mice immunized with FB,-KLH conjugate received three
100-pg doses of immunogen at 2-week intervals. The first injection consisted of 0.1
ml of conjugate in saline mixed with (1 :1 volume ratio) Freund’s complete adjuvant.
The second and third injections consisted of 0.1 ml of conjugate in saline mixed with
(1 :1 volume ratio) Freund’s incomplete adjuvant. Mice immunized with FB,-CT were
injected three times (10-day interval) with 10 pg of immunogen dissolved in 0.2 ml
of PBS. Ether-anesthetized mice were bled from the eye retrobulbar plexus, and
serum was obtained after overnight incubation of blood at 4°C and centrifugation at

1,000 x g for 10 min.

77

 

COCHzCI-lCHzCOOH
H
P t ' 33'”
ro etn ($th (glutaraldehyde)
CHO
COOH
cocrizcncnzcoon

 
 

20

 

22

 

2' CH3 0 N—cr-rz—(CHm -—CH2—N ——Protein

cocuzcr-lcnzcoon H

Figure 3.1. Preparation of fumonisin B, conjugates with glutaraldehyde linkage
via a free amino group (Pestka et al., 1995).

78

ELISA

For antiserum titration, wells of polystyrene microtiter plates (lmmunolon-4-
Removawells) (Dynatech Laboratories, Inc., Chantilly, VA) were coated overnight
at 4°C with 10 pg/ml (100 pllwell) of FB,-OVA, FB,-BSA or FB,-KLH in 0.1 M
sodium carbonate-bicarbonate buffer (pH 9.6). Plates were washed four times with
300 pl of PBS. The wells were blocked for 1 hr at 37°C with 320 pl of 1% (wt/vol)
OVA, BSA or KLH in PBS and then washed four times with 300 pl of PBS. Serially
diluted serum (100 pl) was then added to each well and incubated for 1 hr at 37°C.
Unbound antibodies were removed by washing six times with 0.1% (vol/vol) Tween
20 in PBS (PBS-T), and 100 pl of goat anti-mouse immunoglobulin G peroxidase
conjugate (diluted 1/2000 in 1% OVA-PBS or 1% BSA-PBS) was added to each
well. Plates were incubated for 1 hr at 37°C and washed six times with PBS-T. The
bound peroxidase was determined by the addition of 100 pllwell of TMB substrate
(Sigma Chemical Company) and incubation at 37°C for 15 min. The reaction was
stopped by adding 100 pllwell of 10% H2804. The absorbance at 450nm was
determined with a microtiter plant reader (Molecular Device Corporation, Menlo
Park, CA).

A competitive indirect ELISA (Cl-ELISA) was used to assess the presence
of FB, specific antibodies in mouse sera. Microtiter plates were coated and blocked
as described above. A 50-pl volume of F B, (at various concentrations) dissolved in
PBS was added over FB,-OVA (or FB,-BSA) coated wells, followed by addition of
a 50-pl volume of antiserum (appropriately diluted in 1% OVA-PBS or 1% BSA-
PBS) and incubated at 37°C for 1 hr. Bound antibodies were determined as

79

described above. Relative antibody affinity was arbitrarily designated as the toxin

concentration required to inhibit antibody binding by 50% (I050).

Spleen mRNA Isolation and cDNA synthesis

Total cellular RNA was extracted by homogenizing the murina spleen tissue
of immunized mice in RNA STAT-60 (T EL-TEST Inc., Friendswood, TX). The mRNA
was purified by affinity chromatography on oligo (dT)—cellulose column according to
the manufacturer’s instructions. First-strand cDNA was synthesized from the mRNA
template with Moloney murina leukemia virus reverse transcriptase and random
hexadaoxyribonucleotida [pd(N),,] primers (Phannacia Biotech). Random
hexamers were used because they eliminated the need for lg-spacific primers or
oligo (dT) primers that require synthesis of a long cDNA for obtaining the

sequences encoding the variable heavy or light chain.

PCR amplification and scFv cloning

The variable regions of heavy chain (V,,) and kappa light chain (VK) were
amplified from the first-strand cDNA as described previously (Yuan at al., 1997).
Briefly, the variable regions of heavy chain (V,,) were amplified by using Taq DNA
polymerase and VHBACK and du-VHFOR as primers (T able 2.1). The kappa light
chains (VK) were amplified with dU-VKBACK and VKFOR as primers (T able 2.1). A
93—bp DNA linker (Phannacia Biotech, Piscataway, NJ), containing a sequence
encoding a short flexible peptide, (Gly,Sar)3, was also amplified with LINKBACK

and du-LINKFOR (Table 2.1). A two-step procedure described previously (Yuan at

80

al., 1997) was used for PCR assembly of scFv DNA fragment. The assembled scFv
products were gel-purified and reamplifiad with restriction site tagged primers (RS
Primers Mix) (Pharmacia Biotech) to add an Sfrl site on the 5' and and a Notl site
on the 3' and of the scFv DNA. The scFv DNA products were then digested with Sfil
and Notl restriction enzymes, gel purified and ligated into the phagemid

pCANTAB5E (digested with these same enzymes).

Affinity selection of phage-displayed antl-FB, scFv antibodies

E. coli TG1 competent cells, an amber suppressor strain (supE ), were
transformed by the ligated DNA products above and plated on SOB medium
(Sambrook at al., 1989) containing 100 pg/ml ampicillin and 2% glucose, and
incubated at 30°C ovamight. Colonies were pooled and infected with M13K07
helper phage as described previously (Yuan at al., 1997) to display scFv fusion
protein on the surface of the recombinant phage. The recombinant phages from the
supernatant were filtered through a 0.45 pm-pore-siza filter (Gelman Sciences)

For panning-elution selection, wells of sterile polystyrene lmmulon-4
microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated overnight
(4 °C) with 100 pllwell of FB,-OVA or FB,-BSA or FB,-KLH conjugate (10 pg/ml)
in 0.05 M carbonate-bicarbonate buffer (pH 9.6). The control wells were coated with
the carrier proteins (10 pg/ml) alone. Plates were washed four times by filling each
well with 300 pl of 0.01 M PBS and aspirating the contents. Non-specific binding
was blocked by incubation at 37°C for 1 hr with 300 pl of 1% OVA, BSA or KLH

(wlv) dissolved in PBS and then washing 6 times with 0.05% (v/v) Tween-20 in 0.01

81

M PBS (PBS-T). Recombinant phages (about 10° to 10'° pfulml) were then added
(100 pllwell) The plates were incubated for various times (10 to 60 min) at 37 °C
followed by ten washes with PBS-T and incubation with 300 pllwell of PBS at 37°C
for 1 hr. The wells were then washed another ten times with PBS-T. One hundred
microliter of free FB, (10, 50 and 100 pglml) diluted with PBS was added to the
walls and incubated at 37 °C for 1 hr with rotary shaking (150 rpm) to elute the
bound phages. PBS was used as a control alution reagent. The eluted phages were
collected and passed through a 0.45 pm-pore-siza Iow-protein-binding syringe filter
(Gelman Sciences), and were used to reinfect E. coli TG1 cells for phage titration
and raamplification.

Recombinant phages from selected clones were used to infect E. coli
HB2151 for production of soluble scFv antibodies. Briefly, the infected E. coli
HB2151 cells were cultured in SB medium (35 g/l tryptone, 20 g/l yeast extract, 5
g/l NaCI) supplemented with 100 pglml ampicillin at 30°C with 250 rpm shaking until
they reached an A600 of 0.5. lsopropyl-B-D-thiogaIactopyranoside (IPTG) was then
added to 1 mM final concentration and the cells were incubated on a shaker at 30°C
overnight. Supematants containing the extracellular soluble scFv proteins were
separated from the cell pellets by centrifugation at 1500 x g for 15 min, and filtered
through a 0.45 pm-pore-size filter. The sensitivities and specificities of the scFv
antibodies (phage-associated or soluble forms) were datenninad with ELISA as
described previously (Yuan at al., 1997). Briefly, microtiter plates were coated and
blocked as described above. Next, 50 pl of PB, standard (at various concentrations)

dissolved in PBS was added to the coated wells and simultaneously incubated with

82

50 pl of phage-associated (about 5 x 1010 pfulml in 2x YT medium) or soluble scFv
antibody (various dilutions with 1% OVA-PBS or 1% BSA-PBS or PBS-2% non-fat
dry milk). A monoclonal antibody (diluted 1:20.000) was included for comparison.
The plates were incubated for 1 hr at 37 °C then washed 6 times with PBS-T. The
amount of bound phage-associated scFv antibody was datenninad by the addition
of 100 pl sheep anti-M13—HRP conjugate diluted 122500. The amount of soluble
scFv antibody bound was determined by incubation with 100 pl mouse anti-E tag
(1 pglml) at 37°C for 1 hr followed by washing (six times) and a subsequent
incubation with goat anti-mouse lgG HRP conjugate (diluted 122000). The bound
monoclonal antibody was also detected by the addition of 100 pl goat anti-mouse
lgG HRP conjugate. The plates were incubated for 1 hr at 37°C and then washed
6 times with PBS-T. The amount of anti-M13—HRP and goat anti-mouse IgG HRP
conjugates bound in the walls was assessed by the addition of 100 pl of 3,3',5,5'-
tetramathyI-benzidina (T MB) at 37°C for 15 min. The absorbance was read at 450
nm with a microtiter plate reader (Molecular Devices Corporation, Menlo Park, CA)

after the color development was stopped with 100 pllwell of 10% H2804.

83

RESULTS AND DISCUSSION

Mouse immunization and polyclonal antibodies

Eight mice were immunized and boosted with FB,-KLH immunogen and
another eight mice were injected intraperitoneally with FB,-CT conjugate. Each
mouse was bled one week after every boost and its samm was prepared and
titrated. Titers of the mouse antisera after the second boost were detected in an
indirect ELISA format (Figure 3.2A). The specificity and sensitivity of the antisera
were determined with a competitive indirect ELISA (Figure 3.2B). Concentration of
FB, required for 50% binding inhibition with antisera from FB,-CT immunized mice
(approximately 0.27 pglml) were much lower than that from FB,-KLH immunized
mice (>10 pglml) (Figure 3.2B). Like FB,-BSA conjugate (Azcona-Olivera at al.,
1992a), the conjugate of FB,-KLH was generally ineffective in the production of PB,
antibodies in mice. The apparent high antibody titers in mice immunized with FB,-
KLH might be due to the presence of antibodies that recognize the toxin-carrier
bridge or a conjugate by-product, but which are not inhibited by free F3, in CI-
ELISA. In contrast to immunization with FB,-KLH, immunization with low FB,-CT
doses resulted in the rapid production of PB, specific antibodies with high relative
affinity in all treated animals. Compared with the FB,-KLH immunogen, the
effectiveness of FB,-CT conjugate in the production of FB, antibodies might be

explained on the basis of the CT adjuvant effect (Azcona-Olivera at al., 1992a).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A. B.
-o—FB1-KLH +FB1-CT

E E
til 13
a a
5 E
i i

O 4 I I 1T g g

400 800 1600 3200 6400 0 10 100 1000 10000

Antisera dilution factor (x) Competitive FB1 . nglml
Figure 3.2. Polyclonal anti-FB, antibody production from mice immunized with

FB,-cholera toxin (FB,-CT) and FB,-keyhole limpet hemocyanin (FB,-
KLH) conjugates (Each group had one representative out of eight
mice). Sara were obtained one week after the second boost with the
immunogens. A: Titration of antisera measured in FB,-ovalbumin
(FB,-OVA) conjugate coated Immulon-4 microtiter wells. Bound
mouse antibodies were detected by horseradish peroxidase
conjugated goat anti—mouse IgG. B: Competitive indirect ELISA for
FB, with the antisera (diluted 1:60.000 and 1:3,200 respectively for
FB,-KLH and FB,-CT immunized mice).

85

Construction of phage-display scFv library

Mice immunized with FB,-CT conjugate were sacrificed one week after the
second boost and their spleens were homogenized in RNA STAT-60 (T EL-TEST
Inc.) After mRNA was purified by affinity chromatography on oligo (dT)-cellulose
column, first-strand cDNA was synthesized with Moloney murine leukemia virus
reverse transcriptase and random hexadaoxyribonucleotida [pd(N),,] primers
(Phannacia Biotech). PCR amplification from the mRNA yielded a VH DNA fragment
with a size close to the expected 340 bp (lane 3 in Figure 3.3) and a VK DNA
fragment with a size close to the expected 324 bp (lane 2 in Figure 3.3). A linker
DNA (lane 1 in Figure 3.3) was also produced from a template containing sequence
encoding short peptide (Gly,Ser)4 by PCR amplification with LINKBACK and du-
LINKFOR as primers. The DNA fragments of VK and linker were successfully joined
by PCR assembly (lane 4 in Figure 3.3) and then joined with V,, DNA forming the
scFv DNA fragments (lane 5 in Figure 3.3). After addition with an Sfil site on the 5'
and and a Notl site on the 3' end, the assembled scFv DNA products were digested
with Sfil and Notl restriction enzymes, gel purified and ligated into the phagemid
pCANTAB5E. The ligated products were introduced into E.coIiTG1 competent cells
by chemical transformation. Approximately 10,000 transforrnants were produced
for the construction of scFv phage-display library. Restriction analysis of the
recombinant phagemid DNA isolated from the transformants showed an expected

size of scFv insert DNA in pCANTAB5E.

86

Figure 3.3.

 

PCR amplification of VH and VK genes from mouse splenic mRNA
and the assembly of scFv. Lanes are: (1 ): Linker PCR product; (2):
VK PCR product; (3): VH PCR product; (4): Linker-VK PCR product;
(5):V,,-Linker-VK (scFv) PCR product; Lane M2100-bp ladder DNA
marker (Gibco BRL).

87

Affinity selection of phage-display scFv antibodies

In a preliminary experiment, the newly constructed recombinant scFv phage
library was incubated in FB,-OVA (1 pg/well) coated wells for FB, specific binding
and in OVA coated wells as a negative control of nonspecific binding. After
incubation for 60 min with the scFv phage library and extensive washing with PBS-
T, FB, (100 pglml) or PBS were used to elute the bound phages from the wells.
After five rounds of panning-alution selection, the number of phages eluted by FB,
from FB,-OVA coated wells did not differ significantly (less than one order
difference) from that eluted by PBS. On the other hand. FB, eluted approximately
the same number of phages from both FB,-OVA coated wells and OVA coated
walls. This indicated that FB,-specific scFv phages were not enriched in this
panning-alution selection.

To search for a suitable panning-alution selection procedure for specific
binding and elution. the effects of different FB,-carrier protein conjugates (i.e. FB,-
OVA. FB,-BSA. and FB,-KLH. Figure 3.4 A), scFv phage library incubation time (Le.
60. 20, and 10 min, Figure 3.4 B) and FB, concentration (i.e.10, 50, and 100 pglml,
Figure 3.40) in the elution solution were investigated. No significant enrichment
for FB,-specific scFv phage (phage eluted by FB, from FB,-carrier proteins coated
walls) was found in any condition tested (Figure 3.4). Although the number of
phages eluted by FB, (50, 100 pglml in the elution solution in most cases) from FB,-
OVA or FB,-BSA coated walls was greater than that eluted by PBS. the difference

appeared not due to the presence of FB, specific scFv phages because the number

88

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89

100
Standard procedure was: coating: 1 pg/well of FB,-OVA conjugates

were different conjugates for coating (A), phage incubation time (B).

and FB, concentration for alution (C).

Figure 3.4. Phage titers after panning-alution selection of the phage library.
or OVA alone (in control wells);

of phages (

 

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of phages eluted by FB, from the FB,-carrier protein conjugates coated walls was
similar to that eluted by FB, from wells coated only carrier proteins. Reducing the
phage library incubation time in the coated walls, or decreasing the FB,
concentration in the elution solution did not improve the differentiation between
specific alution (phages eluted by FB, from FB,-conjugate coated wells) and
nonspecific alution (phages eluted by F B, from carrier proteins coated wells) (Figure
3.4).

After failing to enrich F B, specific scFv phages by panning-alution selection.
identification of individual clones with soluble scFv proteins that specifically bound
to FB, was also investigated. Two hundred seventy randomly selected individual
phage plaques from the unselected original scFv phage library and 39 individual
phage plaques selected after four rounds of panning-alution selection (In each
round of selection. phages were incubated in FB,-OVA coated walls for 60 min,
washed, and eluted with 100 pglml FB,) were used to infect E. coli HBZ151 for
soluble scFv protein production. None of the isolates bound FB, specifically in
ELISA.

Although the mice whose splenic mRNAs were used to construct the scFv
phage library in this study produced relatively high affinity antibodies to FB, , no FB,
specific scFv antibody clone has been selected from this scFv phage library.
Sequence changes during the scFv construction procedure and conformation
differences between scFv protein and the native antibodies may cause changes in
specificity and sensitivity for the scFv proteins. These changes may result in the

absence of FB, specific clones in the original scFv phage library. Even if there were

90

a few clones specifically binding to FB, in the library. the high degree of nonspecific
binding of the phage library to the carrier proteins alone plus nonspecific alution
activity by FB, in the panning-alution selection make the enrichment for FB, specific
phages impossible. Considering that phage panning selection also failed to
generate any scFv antibody clone with high affinity to FB, from an scFv phage
library constructed from a hybridoma cell line producing FB, specific monoclonal
antibody (Zhou at al., 1996). an alternative way to clone a functional FB, scFv
antibody is needed. For example, the VH and V,_ can be cloned using PCR-based
RACE (Rapid Amplification of cDNA Ends) technology with primers which are
independent of the variability within the V regions (Ruberti at al., 1994) and with
high fidelity enzymes such as Pfu DNA polymerase. They are then assembled into
an scFv fragment and tested for their activities. The specificity and sensitivity of the
cloned scFv antibody could be improved further by mutagenesis or chain shuffling
(Hoogenboom at al., 1992) or CDR-grafting (Smith at al., 1995; Jung and Pluckthun.
1997)

91

CONCLUSION

Mouse polyclonal antibodies to FB, were readily generated after
immunization with FB,-CT conjugate but not produced with FB,-KLH conjugate
immunization. The relative affinity (measured by FB, concentration required for
50% binding inhibition) of mouse polyclonal antibodies to FB, was 0.27 pglml. From
the splenic cDNA of mice that were immunized with FB,-CT. variable region genes
of heavy- and kappa light-chain were successfully amplified by PCR with previously
designed primers (Yuan at al., 1997). The VH and VK genes were assembled with
a linker DNA to form scFv DNA fragments by PCR and cloned into a phage-display
vector. However. panning-alution selection from this scFv phage-display library did
not result in any enrichment of FB, specific phages. Different FB,-protein
conjugates, phage incubation times, and FB, concentration in the elution solution
were tested for their effects on specific binding and alution. No conditions were
found suitable for FB, specific scFv phage selection in the panning-alution
procedure. The soluble form of scFv protein from approximately 300 randomly
selected individual clones did not show FB, specific binding activity either. An

altamative method is needed to clone functional FB, specific scFv antibodies.

92

PART IV

EXPRESSION OF A FUNCTIONAL ANTI-ZEARALENONE SINGLE-CHAIN FV
ANTIBODY IN TRANSGENIC ARABIDOPSIS PLANTS

93

ABSTRACT
EXPRESSION OF A FUNCTIONAL ANTI-ZEARALENONE SINGLE-CHAIN FV
ANTIBODY IN TRANSGENIC ARABIDOPSIS PLANTS

The efficacy of cloning a recombinant mycotoxin antibody in plants was
tested using Arabidopsis as a model. An anti-zearalenone scFv DNA fragment was
first cloned in the newly constructed phage-display vector (pEY.5) and then
recloned in the plant transformation vector, pKYLX71::35$2. After transformation,
constructs of anti-zearalenone scFv were introduced into immature Arabidopsis
seeds via Agrobacterium tumafacians mediation by vacuum infiltration. Only plants
transforrnad with construct containing a PR-1 b signal peptide sequence produced
transgenic offspring. The anti-zearalenone scFv plantibody from these transgenic
plants bound zearalenone with a high affinity ( leo, 11.2 ng/ml) comparable to
bacteria-produced scFv antibody and the parent monoclonal antibody (mAb) from
hybridoma cell culture. By electron microscopic immunogold labeling, the presence
of anti-zearalenone scFv was mainly detected in the cytoplasm and only
occasionally outside the cell. Like bacteria-produced scFv antibody. anti-
zearalenona scFv plantibody exhibited greater sensitivity to methanol destabilization
than the parent monoclonal antibody did. The sensitivity of anti-zearalenone scFv
plantibody to acidic disassociation was similar to bacterial-produced scFv antibody
and mAb. Expression of specific plantibodies in crops might be useful to neutralize

mycotoxins in animal feeds and to reduce mycotoxin-associated plant diseases.

94

INTRODUCTION

Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecanyl)—l3-rasorcylic acid
lactone] is a mycotoxin produced by members of the genus Fusarium after infection
of corn and small grains ( Mirocha at al., 1971; 1977; Hart at al.. 1982). When fed
to animals. the compound causes hyperestrogenism with symptoms such as
enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse. and
infertility (Hidy at al.. 1977; Mirocha at al.. 1974).

Recently. we developed a single-chain Fv (scFv) antibody with high affinity
to zearalenone (Yuan at al., 1997). Our experience and others (Kramer and Hock,
1996; Dr. Chu in UW, personal communication) showed that scFv assembly from
VH , linker and VK DNA fragments by PCR is one of the most problematic steps in
scFv cloning. The assembly often results in undetectable amplificates or undefined
DNA amplified products with various sizes. To facilitate the cloning and chain
shuffling, and to increase the efficiency of scFv assembly from V,, and VK cDNA
fragments, a new phage-display vector was constructed by replacing a 52-bp Sfi
llNot I fragment in pCANTAB5E (Phannacia Biotech, Piscataway, NJ )with a DNA
fragment encoding the (GIy,Sar)3 linker peptide plus restriction sites which rarely
exist in V gene regions. To confirm the feasibility of the new vector in scFv antibody
cloning. an anti-zearalenone scFv DNA fragment was cloned and expressed with
this new vector in Escherichia coli.

In the last ten years. the expression of specific antibodies or antibody

fragments in plant has attracted great interest (reviewed by Hiatt and Ma, 1992;

95

Conrad an
and has sl
(lavladore
Ansaenkc
mycotoxir
used the
ArabiclOp
zearalen

of a SOIL

Conrad and Fiedler, 1994; Ma and Hein. 1995; Whitelam, 1995; 1996; Smith, 1996)
and has shown some potential in improving plant resistance against pathogens
(T avladoraki at al., 1993) and altering plant metabolic pathways (Owen at al., 1992;
Artsaenko at al.. 1995). To explore the possibility of using plantibodies to neutralize
mycotoxin through passive immunization of animals by feeding. as a first step, we
used the newly cloned anti-zearalenone scFv DNA fragment to transform
Arabidopsis plants. In this report, we demonstrated that the expression of anti-
zaaralenone scFv gene in transgenic Arabidopsis plants leads to the accumulation

of a soluble scFv plantibody with high affinity to the mycotoxin zearalenone.

96

Gen

purt
note

pror

Co

METHODS AND MATERIALS

General

All chemicals and solvents were reagent grade or better. Chemicals were
purchased from Sigma Chemical Company (St. Luis. MO) unless otherwise
noted. All DNA manipulations, if not described, were carried out by standard

procedures (Sambrook at al., 1989).

Construction of scFv cloning vector

A DNA fragment (Pharmacia Biotech, Piscataway, NJ), encoding a short
peptide containing the structurally flexible peptide linker (Gly,Ser)3, was amplified
by polymerase chain reaction (PCR) with Taq DNA polymerase using a sense

primer (5'-TCTATGCGGCCCAGQCGQQQGGCAQTAGTGTCACCGTC-3')
containing Sfi l and Spa I restriction sites (undaflined) and an antisense primer
(5'-AGCACCT§C§§CC§QCTGAGTGAGQTQGATGTCCGATCC—S') containing Not
I and Sec I sites (underlined). After electrophoresis in 1.5% low melting point
aQarosa gal (Boehringar Mannheim Biochemicals, Indianapolis, IN), the amplified
fr’agment was excised from the gel and purified with QIAEXII gel extraction kit
(Q IAGEN. Chatsworth, CA). The purified DNA fragment was then digested with 817
I and Not I and ligated into the same enzyme digested pCANTAB5E (Pharmacla

Biotech). creating pEY.5 (Figure 4.1).

97

Ci‘i

Flc

Sac I (2404)

   
   
  
 

so: (2318?” I (2332' Nil (2416 BspE 1 (2442)
A Hind m (2235) m . Stop Codon (2470)
' PM I (2233) Barn 1 (2578)

All III (1878)

  
  
  

a 1 (3359)
pEY.5 "a” ' (3555’
(4565 bp)
EcoRI (3689)
Natl (3850)
Dre m (4126)
S“ | (505) BMAI(4129)
B. Xho I (319) “5177251?“ (7)
2314
I
60W GGC AQT AQT GTC ACC GTC TCC TCA GGT GGA

GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC
2424

l
GAG CTC ACT CAG GCG GCC GCA E tag-Amber stop codon -fd gene 3

 

Figure 4.1 . Map of phage-display vector pEY.5. (A). Restriction map with unique
restriction sites marked. (B). The nucleotide sequence of linker (bold
sequence) and the VH and VK cloning sites (underlined) in pEY.5.

98

Anti-1 eara

VH _,
clone pQY
attac led p
TGA GGA,
AT( rAC ‘
GSI GTC
for ‘ 'K DI\
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DN time
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| 3 Id Ilg
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Cl 'nSII

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Anti-zearalenone scFv antibody cloning in pEY.5
VH and VK DNA fragments were ra-amplifiad from an anti-zearalenone scFv
clone pQY1.5 phagemid DNA (Yuan at al.. 1997) using the following restriction site
attached primers. The primers used for VH DNA re-amplification were: VHFORY (5'-
TGAGGAGACGGTGACACTAQTGCCTTGGC-3') and VHBACKY (5'-
ATGACTCGCGGCCQAGQQGGCCATGGCCSAGGTSMARCTGCA
GSAGTCWGG-3'); S= C or G; M=A or C; R=A or G; W=A or T. The primers used
for VK DNA ra-amplification were: VKBACKY (5'-TCGGACATC§A§CTQACTCA
GTCTCC-3') and VKFORY (5'-GAGTCA‘I'I'CT§CGQCQGCCCG‘ITI’BAKYT C
CARCTTKGTSCC-3'); B=T or C or G; K=T or G; Y=C or T. The re-amplified VH
DNA fragments were digested with Sfi l and Spa l and ligated into pEY.5 digested
with the same enzymes. The ligated products were then digested with Sac I and Not
I and ligated with the same enzyme digested VK re-amplified DNA fragments.
yielding pEY.5HL. The pEY.5HL phagemid DNA was introduced into E. coIiTG1 cell
forthe production of recombinant phages in the presence of helper phage M13KO7
and into E. coli HB2151 for soluble scFv antibody production as described
previously (Yuan et al., 1997). The soluble scFv antibody from E. coli cultures was

Characterized with ELISA as described (Yuan at al.. 1997).

construction of plant scFv expression plasmids
The anti-zearalenone scFv DNA fragment was amplified from a zearalenone
biriding positive clone, pEY.5HL3 (reconstructed in pEY.5 from pQY1.5), by

DOIymerase chain reaction (PCR) using Pfu DNA polymerase (Stratagane. La Jolie.

99

CA )
GTG
CCC
amp
with
yielc
frorr
cree
HITII
sigr
seq
p81
Fin
wa
cre
Dl—

m;

PI;

it;
Be

ar

CA ) with the following primers: Sense primer ( 5'-TAT&_C§_GTATGGCCCAG
GTGAAACTGC-3') containing an Sst ll site and antisense primer (5'-
CCCTQTAGACCAGACGTTAGTAAATGAA—S') containing an Xba I site. The
amplified DNA fragment was isolated from a low melting point agarose gel. purified
with QIAEXII, digested with Xba I. and ligated into Hinc lI/Xba l -digested pUC19,
yielding pQY2. For expression without a signal peptide. 3 Hind Ill-Xba I fragment
from pQY2 was ligated into the Hind Ill-Xba l sites of pKYLX71::35S2 directly,
creating pQY4.1-1. For expression with a plant protein signal sequence. a 110-bp
Hind Ill/Sst ll fragment containing tobacco pathogenesis-related protein PR-1b
signal peptide (sp) sequence from pSlG3. a clone with the PR-1b signal peptide
sequence (Gopalan at al., 1996) ligated in the EcoR V site of Stratagene’s
pBIuescript SK, was ligated into the Hind Ill-Sst ll sites of pQY2. generating pQY3.
F inally, a Hind Ill-Xba I fragment containing the sp-scFv fusion fragment from pQY3
was ligated into the Hind III-Xba I sites of pKYLX71::3582 (Maiti at al., 1993),
creating pQY4.4-1. Both pQY4.1-1 and pQY4.4-1 were introduced from E. coli
DHSa into Agrobacterium tumefacians strain GV3850 (Zambryski at al., 1983) by

triparental mating (Figurski and Heliniski, 1979).

P lant transformation

Arabidopsis thaliana acotype Columbia (Col. GLABROUS1) plants were
transformed using a vacuum infiltration protocol similar to the one described by
Bechtold at al. (1993). Mature seeds from these infiltrated plants were germinated

and selected on plant nutrient agar plates (Bechtold at al., 1993) supplemented with

100

50 pglml kanamycin. Kanamycin resistant seedlings were transferred into soil and

leaves from two-month-old kanamycin resistant plants were tested for scFv antibody

binding activity with ELISA.

Expression of scFv plantibody and ELISA analysis

lmmulon-4 microtiter walls (Dynatech Laboratories, Inc., Chantilly, VA) were
coated with 1 pglwall of zearalenone-OVA (ZEN-OVA) conjugate in 100 pl 0.05M
carbonate-bicarbonate coating buffer (pH9.4) at 4°C overnight. The wells were
washed and blocked as described previously (Yuan at al.. 1997). Two to three
leaves from two-month-old transgenic plants were cut and ground in a 1.5 ml
microcentrifuge tube on ice with a pestle. After centrifugation at 13,000 rpm in a
microcentrifuge for 10 min at 4°C, the sap (supernatant) was collected. Fifty
microliters of the leaf sap was diluted 1:1 (vol/vol) with 2% non-fat dry milk in PBS
and added to each well, followed by incubation at 37°C for 1 hr. After washing 6
times with 320 pllwell of PBS-0.1% Tween 20, 100 pllwell of mouse anti-E tag
antibody (1 pglml) was added. followed by addition of the goat anti-mouse lgG HRP
conjugate (diluted 1:2000 in 2% non-fat dry milk in PBS). Finally, 100 pllwell of TMB
substrate (Sigma Chemical Company) was added and incubated at 37°C for 1 5 min.
The reaction was stopped by adding 100 pllwell of 10% H2804. The absorbances
at 450nm were determined with a microtiter plant reader (Molecular Device
Corporation, Menlo Park, CA).

For competitive ELISA, 50 pllwell of free zearalenone at various

concentrations (0. 1. 10, 100, and 1000 ng/ml in PBS containing 1% methanol) was

101

adr
(frc

bor

SCI
09
an
pl-
inc
ba
KL

WE

Pr

pr

Di

added into zearalenone-OVA coated wells. then 50 pllwell of diluted (1 :49) plant sap
(from GV3850/pQY4.4-1 transformed plants) was added. After 1 hr incubation. the
bound plantibodies were detected in the same way as described above. For
comparison. a CI-ELISA with diluted (1 :9) bacterial anti-zearalenone scFv
(produced from E. coli HBZ151/pEY.5HL3) was performed simultaneously.
Methanol effects on the binding activity were measured for mAb. bacterial
scFv antibody and scFv plantibody ( diluted 10 folds in 10 % non-fat milk-PBS) as
described previously (Yuan at al., 1997). The effect of the pH environment on these
antibodies was also tested. The antibodies were diluted (1 :5 vol/vol) in pH 2.3 and
pH 6.3 (adjusted with 0.05 M citric acid and 0.05 M NazHPO, ) buffer solution, and
incubated at 4°C, overnight. A portion of the pH 2.3 treated sample was adjusted
back to pH 6.3 and incubated at room temperature for one hour. To zearalenone-
KLH conjugate (1 pglwall) coated walls, 100 pllwell of pH treated antibody samples

was added. Bound antibodies were determined as described above.

Protein analysis

Proteins in plant leaf sap from independent transgenic plants were separated
in a 12% SDS-polyacrylamide gel. Two identical gels were run at the same time.
After electrophoresis, one gel was stained in Coomassie brilliant blue G-250. The
proteins in another gel were transferred to a nitrocellulose membrane (Schleicher
& Schuell, Keene, NH). Mouse anti-E tag antibody, goat anti-mouse IgG alkaline

phosphatase conjugate (Sigma Chemical Company, St. Louis. MO) and NBT/BCIP

102

substrate

similar to

 

ScFv an
S
transgen
glutarald
tempera
1% osiu
followed
embedcl

Ultrathir

 

substrate (Pierce, Rockford. IL) were used to detect plantibodies in a procedure

similar to that previously described (Yuan at al., 1997).

ScFv antibody cytolocation with immunogold labeling

Segments ( approximate 0.5 x 1.0 mm) of leaves from 2-month-old
transgenic Arabidopsis plants were fixed by vacuum infiltration in 2%
glutaraldelhyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 hr at room
temperature. After 3 washes in the above buffer, the samples were post-fixed with
1% osium tatroxide in 0.1 M sodium cacodylate buffer at room temperature for 2 hr,
followed by dehydration with a graded acetone series. The samples were then
embedded by infiltration with Resin “Spurtol” (Spurr, 1969; Kushida, 1974).
Ultrathin sections were cut and supported on formvar-coated grids.

The grids containing ultrathin sections were pretreated by incubation in a
saturated aqueous solution of sodium metaperiodata as described by Bendayan
and Zollinger (1983) After washed throughly in distilled water, the pretreated grids
were blocked for 1 hr at room temperature in 20 mM Tris-buffered saline (T BS, pH
7.5). containing 0.05 % Tween 20 . 1 % wt/vol bovine serum albumin and 0.01 %
NaN3. The samples on the grids were then incubated with the primary antibody
(mouse anti-E tag antibody) diluted in the blocking buffer for 2 hr at room
temperature. In the case of testing for the cross-reactivity of anti-zearalenone scFv
antibody to Arabidopsis. samples from non-transformed control plants were
incubated with anti-zearalenone scFv antibody first, then with mouse anti-E tag

antibody. After four 5 min washings with TBS. the grids were incubated with goat

103

anti
tem
dist
rins
al.,
anc
Nel

the

anti-mouse lgG gold conjugate (10 nm gold particle) (1: 20 dilution) for 2 hr at room
temperature. After four 5 min washings with TBS and two 5 min washings in
distilled water, the grids were post-stained with 1 % uranyl acetate for 30 min,
rinsed in distilled water, and then stained with Hanaichi’s lead solution (Hanaichi at
al., 1986) for 5 min. The grids were then rinsed in 20 mM NaOH and distilled water
and examined with a CM10 Transmission Electron Microscope (Philips, The
Netherlands). Immunogold labeling of sections of non-transformed Arabidopsis

thaliana ecotypa Columbia was performed as a control.

104

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expn
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RESULTS

Cloning of anti-zearalenone scFv In pEY.5

To determine the feasibility of cloning vector pEY.5 for scFv antibody
expression. the DNA fragments of VH and VK from pQY1.5 (Yuan at al., 1997) were
amplified with the above primers and ra-cloned into pEY.5. In E. coli HB2151 , one
new clone (pEY.5HL3) produced soluble scFv antibody with high affinity to
zearalenone (Figure 4. 2). The IC50. concentration of free zearalenone required for
50% inhibition of binding, was 15.5 nglml, similar to that previously described for
pQY1.5. Either V,, or VK alone cloned in pEY.5 is in frame with E-tag and 93p,
therefore this vector is also suitable for selecting single domain Fv antibodies and
chain shuffling. However, either V,, or VK alone from pQY1.5 (Yuan at al.. 1997) did
not show any zearalenone binding activity when they were cloned into pEY.5
separately (data not shown). indicating that both VH and VK were important for the
scFv binding to zearalenone. It was notable that E.coli HB2151/pEY.5HL3 grew
much slower than HB2151/pEY.5, which suggests the functional anti-zearalenone

scFv expression may affect the growth of the E. coli host cells.

105

100
90
80
70
60
50
4O
3O
20
10

 

 
    

-o—EY.5HL3
+Plantibody

Inhibition of binding, %

 

 

I
I

O 1 10 100 1000

Zearalenone, ng/mL

Figure 4.2. Sensitivity and specificity of anti-zearalenone scFv antibodies
measured by competitive indirect ELISA using zearalenone-
ovalbumin coated wells. Bound soluble scFv antibody was detected
by mouse anti-E tag antibody, followed by horseradish peroxidase
conjugated goat anti-mouse lgG. EY.5HL3 was produced in E. coli
and plantibody was produced in Arabidopsis.

106

Expression of anti-zearalenone scFv plantibody

The anti-zearalenone scFv DNA fragment from pEY.5HL3 was re-cloned into
a plant transformation vector to generate pQY4.1-1 and pQY4.4-1(Figure 4.3).
About 1% of 20,000 seeds from plants transformed with GV3850/pQY4.4-1 were
kanamycin resistant. whereas plants transformed with GV3850/pQY4.1-1 produced
no kanamycin resistant seedlings from approximately 20,000 seeds (Figure 4.4).
In the putative transgenic plants (kanamycin resistant) transformed with
GV3850/pQY4.4-1. approximately 20% of the tested plants produced functional
antibody for zearalenone as determined by ELISA. Western analysis showed that
GV3850/pQY4.4-1 transformed Arabidopsis plants produced a 32-KDA scFv
plantibody protein (Figure 4.5), similar in size to the scFv antibody produced in E.
coli (Yuan et al., 1997). The specificity and sensitivity to zearalenone of the
plantibody was determined by CI-ELISA (Figure 4.2). The plantibody had an IC50 of
11.2 ng/mI for zearalenone, showing similar affinity to zearalenone as the scFv
antibody produced from bacteria and monoclonal antibody from hybridoma cell
culture (Yuan at al., 1997).

From each of the T0 transgenic Arabidopsis plants, 15 to 20 T, offspring
seedlings were tested with ELISA for zearalenone binding. Some plants had an
approximately 1 :1 segregation ratio of functional anti-zearalenone scFv plantibody
producing offspring to non-producing offspring; others had a 1:0 segregation ratio.
This suggests that both single and double integrations of plant chromosomes by the

anti-zearalenone scFv DNA fragment occurred. In the T2 generation, all plants

107

Figure 4.3.

Hind
35Sz

 

P...

 

 

A A A A A A A A A A A A A A

9 9

Anti-zearalenone scFv E-tag

III Sst II Xba I

((666

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sp Tu Tu:
32553252223552isisisizisibfl H I

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1c:

 

Anti-zearalenone scFv E-tag

The transgene in plant transformation constructs pQY4.1-1 (A) and
pQY4.4-1 (B). 3582: Can 35S promoter with a duplicated enhancer;
sp: plant pathogenicity-related protein PR-1 b signal peptide
sequence; Tim and Tm: transcription termination sequences for rch
and nos respectively; Pm: nos promoter; Kn': kanamycin resistance

gene.

108

 

Figure 4.4.

The germination of Arabidopsis tansformant seeds on kanamycin
selection medium plates. A. Seeds from un-transformed control
plants; B. Seeds from plants transformed with pQY4.4-1, targeting
scFv antibody to the apoplastic space with signal peptide sequence.
C. Seeds from plants transformed with pQY4.1-1, targeting scFv
antibody to the cytoplasm.

109

Figure 4.5.

 

SDS-polyacrylamide gel electrophoresis and Western blot analysis of
anti-zearalenone scFv plantibody expression. Soluble proteins of
Arabidopsis from un-transformed plants (lane 1), vector
pKYLX71::35$2 transformed plants (lane 2), and pQY4.4-1
transformed plants (lane 3) were subjected to SDS-PAGE and stained
with Coomassie brilliant blue G-250 (A). Western blot of duplicate
SDS-polyacrylamida gal detected by anti-E tag antibody (B). Lane M:
protein molecular size marker with 10KDa-ladder.

110

produced anti-zearalenone scFv plantibody without segregation. This indicates that

the anti-zearalenone scFv antibody gene was stably integrated into the genome of

Arabidopsis.

The effects of methanol and low pH on plantibody

As shown previously. anti-zearalenone scFv antibody from bacteria exhibited
greater sensitivity to methanol destabilization than the parent monoclonal antibody
(Yuan at al., 1997). To test the effects of post-translational modifications on the
stability of scFv antibodies, anti-zearalenone scFv plantibody was compared with
scFv antibody produced in E. coli and the parental monoclonal antibody for their
stabilities in methanol and in acidic environment. The scFv plantibody from pQY4.4-
1 transformed Arabidopsis plants was also shown very sensitive to methanol
destabilization (Figure 4.6), whereas the parent monoclonal antibody exhibited
tolerance to much higher concentrations of methanol. In the low pH environment
(pH 2.3), however, all three antibodies (monoclonal antibody. bacterial scFv
antibody and scFv plantibody) lost binding activity to zearalenone (T able 4.1).

When the pH was adjusted back to neutral (pH 6.3), the binding activity recovered.

111

 

 
   
 

 

 

 

E 1.6 -
O
z;
*5 1.2 *-
0
§ -D-mAb
“E 0.8 ~~ —o—EY.5HL3
§ +Plantibody

0.4 ~—

0 I l l I l I 7

Methanol in the well, %

Figure 4.6. Effect of methanol on the binding of anti-zearalenone monoclonal
antibody (mAb) and bacterial scFv antibody (EY.5HL3) and plant scFv
antibody (plantibody) to immobilized zearalenone-KLH conjugate.

Table 4.1. Effect of pH environment on antibody binding activity.

 

 

 

 

 

 

 

Anti-zearalenone Relative binding activity in different pH environment, %*
antibody pH 6.3 pH 2.3 pH 2.3 than pH 6.3
mAb 100 0 99
Bacterial scFv 100 0 98
scFv plantibody 100 0 89

 

 

 

 

*: The binding activity at pH 6.3 was set as 100%.

112

Localization of anti-zearalenone scFv plantibody

Transgenic Arabidopsis plants expressing anti-zearalenone scFv antibody
were processed and labeled for electron microscopic immunolocalization as
described in materials and methods. Gold particle deposition indicate the presence
of scFv antibody mainly in the cytoplasm with a few labeling signal in the
intercellular spaces (Figure 6A,B). Deposition was not found in the non-transformed
control plant samples (Figure 4.70). However. incubation of non-transformed plant
sections with the anti-zearalenone scFv antibody than with mouse anti-E tag
antibody followed by goat anti-mouse lgG gold conjugate caused many labeling
signals evenly distributed in the cytoplasm (Figure 4.7C). indicating that anti-

zearalenone scFv antibody can bind to Arabidopsis cell components.

113

 

Figure 4.7.

Cytolocation of anti-zearalenone scFv plantibody by immunogold
labeling and transmission electron microscopy. Leaf sections from
plants expressing anti-zearalenone scFv plantibodies (A, B) and non-
transfonned control plants (D) were detected by mouse anti-E tag
antibody followed by goat anti-mouse lgG gold conjugate. (C): Leaf
sections from non-transformed control plants were pre-incubated with
anti-zearalenone scFv antibody. than with mouse anti-E tag antibody
followed by goat anti-mouse IgG gold conjugate. CW, cell wall; CY,
cytoplasm; ICS, intercellular space; Arrows: gold labeling;
Magnification x 39,000.

114

DISCUSSION

In this investigation, an scFv plantibody with high affinity to zearalenone was
produced from stable transgenic Arabidopsis plants . To our knowledge, this is the
first report of an anti-mycotoxin antibody gene expressed in plants. Like other scFv
plantibodies (Bruyns at al.. 1996), this anti-zearalenone scFv plantibody had similar
antigen-binding properties as bacteria-produced scFv antibody.

The anti-zearalenone scFv DNA fragment was first cloned in a newly
constructed vector. This new vector, pEY.5, maintains the advantageous features
of the original vector pCANTAB5E , such as an inducible lac promoter, a widely
used linker (Gly,Ser)3, a highly specific E-tag, an amber stop codon allowing the
switch between expression of membrane anchored scFv-93p fusion and soluble
scFv simply by changing the expression host. It also allows the direct cloning of VH
and VK, with the introduction of two rare cutting restriction enzyme sites (Spa l and
Sac | ). In the Kabat Database of Sequences of Proteins of Immunological Interest
(http://immuno.bme.nwu.edu). the Spa I recognition sequence was found in less
than 1% of 4701 known murine VH chains and 1% of 2492 known murine VK chains.
Sac I has a slightly higher cutting frequency, but accounts for only 4.5% and 2.3%
of known V,, and V, chains, respectively. This indicates that the majority of variable
region cDNAs will be free from intemal cutting. We tested the feasibility of this new
vector by cloning and expressing an anti-zearalenone scFv in E. coli. By utilizing the

Sfi l/Spa l or Sac l/Not I sites. this new vector will also facilitate chain shuffling.

115

which will be useful for cloning compatible variable chains and improving antigen
binding ability (Kang at al., 1991; Marks at al., 1992a).

For plant transformation, two constructs of the anti-zearalenone scFv
were made by fusion with or without the N-tenninal signal peptide from tobacco
pathogenesis related protein PR-1 b, which has been used to secrete foreign
proteins to the plant apoplast (Dixon at al., 1991; Lund and Dunsmuir, 1992). The
PR-1b signal sequence was included in the expectation that it would target the
fusion protein to the endoplasmic reticulum where the signal peptide would be
cleaved and the scFv protein transported to the apoplast via the default pathway
(Danecke at al., 1990). Plants transfonnad with this signal sequence containing
construct produced many transfonnants expressing scFv plantibody with high
affinity to zearalenone. However, electron microscopic immunolocalization indicated
the presence of the scFv plantibody mainly in the cytoplasm. Only low level of
labeling signals existed in the intercellular space and was limited to those parts of
intercellular space that were filled with electron-dense material. The labeling signals
in the cytoplasm might reflect a binding activity of anti-zearalenone scFv to some
plant cell components. As a matter of fact, immunogold labeling showed that antl-
zearalanone scFv antibody binds to non-transformed Arabidopsis cell components
in the cytoplasm. Although some antibodies or antibody fragments fused with plant
signal sequences have been confirmed by immunogold electron microscopy to be
secreted through the cell wall into intercellular spaces. despite limitations in cell wall
porosity (De Wilde at al., 1996), others were retained in endoplasmic reticulum

compartment or in the cytoplasm (Dllring at al.. 1990; Artsaenko at al., 1995;

116

Schouten at al., 1997). The construct without signal sequence failed to generate
any transgenic plants in this study. One explanation might be that high
accumulation of anti-zearalenone scFv in the plant cytoplasm and the sequential
binding to plant cell components was toxic to the host plant cell.

As previously reported. anti-zearalenone scFv antibody produced from
bacteria had greater sensitivity to methanol destabilization than its parent mAb. One
of the possibilities may be the differences between prokaryota and eukaryote in
protein post-translational modifications, especially glycosylation. N—linked
glycosylation of proteins in plants is very similar to the glycosylation process in
mammals (Jones and Robison. 1989; Sturm at al., 1987). Possible plant post-
translational changes in addition to glycosylation include fatty acylation.
phosphorylation. sulfation. or modifications that change the pl of the protein (Jonas
and Robinson, 1989). The comparison between antibody produced in bacteria and
plants may help elucidate the role of post-translational modifications in stabilizing
antibody. Unfortunately. it was not possible to test the hypothesis that differences
between anti-zearalenone scFv antibody and its parent mAB in methanol sensitivity
reflect structural differences derived from post-translational modifications because
the anti-zearalenone scFv lacks the canonical Asn-X-Ser/Thr signal sequence for
glycosylation.

Some mycotoxins not only cause toxic effects to animal and humans, but
also serve as virulence factors for the producing fungi in causing plant diseases.
The mycotoxin zearalenone has been reported to cause certain phytotoxicities

(Wakulinski, 1989; Vianello and Macri, 1981; Nedalnik. 1993). The expression of

117

anti-zearalenone antibody in plants might be useful to reduce the severity of plant
diseases associated with this mycotoxin.

Antibodies have been used successfully as antidotes for some low molecular
weight toxins through passive immunization (reviewed by Baud at al., 1997).
However, large quantities of antibody are generally required for passive
immunization in vivo. The prospect of harvesting antibody on an agricultural scale
offers the potential for economic production of almost limitless amounts of antibody.
The production of antibody in edible plants makes it possible to deliver large amount
of toxin specific antibody through food and feed.

Feed-delivered antibody could theoretically bind zearalenone in the intestine
providing it can survive passage through the acidic stomach environment. When
the anti-zearalenone antibody was subjected to low pH. the mAb and scFv
antibody from both bacteria and plants lost their binding ability. However, all three
antibodies recovered their binding activity when the pH was adjusted back to neutral
(pH 6.3). This suggests that the pH affects on the binding activity is not irreversible
denaturation. Thus. providing these antibodies survive the proteolysis in the gut,
they could recover binding to zearalenone in the small intestine, where the pH is
close to neutral. Whether the anti-zearalenone plantibody reported in this study will
neutralize zearalenone in animal feed and reduce toxicity will be determined in the
future.

The expression of mycotoxin specific plantibody may also be useful in
reducing mycotoxin-associated plant diseases. Especially, the catalytic plantibodies

expressed in crops may degrade mycotoxins when they are produced.

118

PART V

IDENTIFICATION OF MIMOTOPE PEPTIDES WHICH BIND TO THE
MYCOTOXIN DEOXYNIVALENOL-SPECIFIC MONOCLONAL ANTIBODY

119

ABSTRACT

IDENTIFICATION OF MIMOTOPE PEPTIDES WHICH BIND TO THE
MYCOTOXIN DEOXYNIVALENOL-SPECIFIC MONOCLONAL ANTIBODY

A monoclonal antibody (mAb) 6F5 that recognizes the mycotoxin
deoxynivalenol (DON. vomitoxin) was used to select for peptides that mimic the
mycotoxin by employing a library of filamentous phage which display random 7-mar
peptides on their surface. Two phage clones were selected from the random peptide
phage-display library and found to code for the amino acid sequences SWGPFPF
and SWGPLPF. These clones were named DONPEP.2 and DONPEP.12,
respectively. A competitive enzyme-linked immunosorbent assay (ELISA) strongly
suggested that these two phage-displayed peptides bound to mAb 6F5 specifically
in the deoxynivalenol binding site. The amino acid sequence of DONPEP.2 plus a
structurally flexible linker at the C-terminus. i.e. SWGPFPFGGGSC. was
synthesized and tested for its ability to bind to mAb 6F5. This synthetic peptide
(C430) and deoxynivalenol competed with each other for mAb 6F5 binding. When
translationally fused with bacterial alkaline phosphatase. DONPEP.2 bound
specifically to mAb 6F5 while retaining enzyme activity. The potential use of these
peptides as immunochemical reagents in deoxynivalenol immunoassay was
evaluated with a deoxynivalenol-spiked wheat extract. When peptide C430 was
conjugated to bovine serum albumin, it elicited antibodies in mice and rabbits that
were specific only to the peptide C430 but not to deoxynivalenol. In an in vitro
translation system containing rabbit reticulocyte lysate. the synthetic peptide C430

did not inhibit protein synthesis but did show antagonism toward deoxynivalenol-

120

induced protein synthesis inhibition. These data suggest that the peptides selected
in this study bind to both mAb 6F5 and to the ribosoma at the same sites as

deoxynivalenol does, but possibly in a slighter different orientation.

121

INTRODUCTION

Deoxynivalanol (DON, vomitoxin, Figura1.3) is one of the sesquiterpene
mycotoxins classified as 12,13-epoxy-trichothecenas (Mirocha at al.. 1977). It
occurs naturally in infected corn (Hart at al.. 1982; Mirocha at al.. 1976), small
grains (Hart and Braselton, 1983; Naish and Cohen, 1981), and mixed feeds
(Mirocha at al., 1976). DON is mainly produced by the fungus Gibbaralla zaae
(Schwein) Patch (anamorph =Fusarium graminaarum Schwabe). At the cellular
level, the main toxic effect of DON is inhibition of protein synthesis via binding to the
ribosome and by interfering with peptidyltransferase (Ueno, 1983; Thompson and
Wannemacher, 1986; Betina, 1989b). In animals. DON can cause anorexia and
emesis (vomiting) (Mirocha at al., 1976; Rotter at al.. 1996); Other toxic effects of
DON include skin irritation, hemonhaging. hematological changes, human
lymphocyte blastogenesis impairment. radiomimetic effects, apoptosis (cytotoxicity)
and immunotoxicity (Ueno, 1983; Forsell and Pestka. 1985; Forsell at al.. 1986;
Pestka at al.. 1987; Pestka at al., 1994b; Pestka and Bondy, 1994; Ueno at al.,
1995; Rotter et al., 1996).

A major means for eliminating DON from human and animal food. like for
most other mycotoxins. is to detect and divert contaminated raw materials from feed
and finished food use. Conventional methods for DON detection (Pathre and
Mirocha. 1977; Scott, 1982). including gas chromatography, gas chromatography-
mass spectrometry, high-pressure liquid chromatography, and thin-layer

ch romatography. involve considerable sample preparation, are time-consuming. and

122

require technical expertise. Compared with other methods, immunoassays have
several advantages for rapid field tests, including high specificity, sensitivity, facile
sample preparation, and ease of use (Pestka at al., 1995). Following the
development of a reliable monoclonal antibody to DON (Casale at al., 1988),
immunological methods, mainly enzyme-linked immunosorbent assay (ELISA). have
been used widely for detection of DON (Pestka at al.. 1995). Such developments
have led to a great demand for specific antibodies and related immunochemical
reagents for the assay. However. chemical conjugation of DON to a carrier protein
or an enzyme has low efficiency because it involves extensive modification and
blocking stages and causes substantial bridge-group interferences and un-wanted
cross-reactions (Casale et al., 1988; Wilkinson at al., 1992; Xiao at al., 1995; Pestka
at al.. 1995). Secondly, when DON is conjugated to a carrier protein. it is weakly
immunogenic. Finally. since DON is toxic and is included as standard and
conjugate in immunoassay. it potentially poses a toxicity risk to the kit user.

A possible altamative to the use of mycotoxins as immunochemical reagents
would be to develop protein or peptide mimics that serve the same function. One
approach for this is through generation of anti-idiotypa antibodies (Chanh at al.,
1989; 1990; 1991;1992; Hsu and Chu, 1994; Chu at al.. 1995). which mimic the
surface structure of low-molecular-weight biological toxins. Sometimes the
sensitivity of original antibody can be improved via generation anti-anti-idiotype
antibody (Chanh at al., 1990). However. these techniques are time consuming and
costly. A new technique, namely phage display, has been developed more recently.

In this procedure, foreign peptides and proteins can be genetically fused to the N-

123

terminus of the minor coat protein 93p of the filamentous phage fd. resulting in
display of the peptides and proteins on the surface of the virion (Smith. 1985). By
insertion of random oligonucleotide sequences into the 93 gene of filamentous
phage fd. phage display provides a means of constructing extensive peptide
libraries that may be screened to select peptides with specific affinities or activities
(Cwirla et al., 1990; Devlin et al., 1990; Scott and Smith, 1990). Phage-displayed
short peptide libraries have been widely used in a number of applications (Cortese
at al.. 1995; Lowman, 1997; Katz, 1997). including epitope mapping (Scott and
Smith. 1990; Cwirla at al., 1990), mapping of protein-protein contacts (Hong and
Boulanger, 1995), identification of protease substrate (Smith at al., 1995).
identification of integrin (O’Neil at al., 1992) and other receptors (Doorbar and
Winter, 1994), antagonists (Pierce at al., 1996), and ligands (Katz, 1997). Few
studies, however, have used this technology to select peptide mimics of non-
proteinaceous chemicals with the exception of Iectin (Oldenburg at al., 1992; Scott
at al., 1992), biotin (Weber et al., 1992). and carbohydrate (Hoess at al., 1993).
Given the feasibility of selecting peptides that mimic non-proteinaceous
chemicals, we were interested in determining whether peptide mimics of low-
molecular-weight mycotoxins could be identified. Once selected, these peptide
mimics might be useful in mycotoxin immunoassay and for development of new
antibodies against mycotoxins. Here we describe two closely related heptapeptide
mimotopes for DON selected from a phage-display random peptide library and their

application to the analysis of DON by ELISA. The mimotope peptide was also

124

compared with DON for cytotoxicity in cell culture and in vitro protein synthesis

inhibition.

125

MATERIALS AND METHODS

Reagents

All inorganic chemicals and organic solvents were reagent grade or better.
Ovalbumin (OVA) (crude), bovine serum albumin (BSA)(fraction V), 3,3',5,5'-
tetramethyl-benzidine (TMB), phenylmethylsulfonyl fluoride (PMSF).
polyoxyethylana sorbitan monolaurata (Tween 20). deoxynivalenol (DON). mouse
immunoglobulin G. and goat anti-mouse IgG peroxidase conjugate were purchased
from Sigma Chemical Co. (St. Louis, MO). Dimethyl sulfoxide (DMSO) and
methanol were purchased from J. T. Baker Inc. (Phillipsburg, NJ). Instant nonfat dry
milk ( Commander Foods, Inc., Syracuse. NY) was purchased from a local grocery
store. DON-BSA, DON-OVA, and DON-horseradish peroxidase (DON-HRP)
conjugates at DON’s 3-hydroxyl group were made though an activated N-
hydroxysuccinimide ester as described by Casale at al. (1988) (Figure 5.1) and
kindly provided by Frank E. Klein (Neogen Corporation, Lansing, MI). Sheep anti-
M13 horseradish peroxidase (HRP) conjugate was purchased from Phannacia
Biotech Inc. (Piscataway, NJ). m-MaleimidobenzoyI-N-hydroxysulfosuccinimide
ester (Sulfo-MBS) and p-nitrophenyl phosphate disodium salt tablets (pNPP) was
purchased from Pierce Chemical Company(Rockford. IL). Anti-DON monoclonal
antibody from hybridoma cell line 6F5 was prepared as described previously
(Casale at al., 1988).

126

 

H3C

  

  

 

 

 

 

. his)
0 CH3
0

H9C4._\B/

/

L---0—C(CHZ)2C —-OH

DON-boronate

||
0

 

i PM
0 CH3
0

H9C4_\B/

DON-boronate-HS

 

 

0—C(CH2)2C —OH

     

 

 

l
i f”3l
0” CH3
OH

DON-HS

l

....o—c(cnz)2cl —Protein

 

 

Figure 5.1.

 

DON-HS-Protein conjugate

Preparation of deoxynivalenol (DON) conjugates via 3-hydroxyl

group (Pestka at al., 1995).

127

Phage-display peptide library and amplification

A phage-display heptapeptide library with 2 x 10" sequence complexity
(random peptide 7-mers fused to the minor coat protein 93p of the filamentous
coliphage M13) was purchased from New England Biolabs. Inc. (Beverly, MA). The
library was amplified by adding 5 pi (= 1011 pfu) recombinant M13 phage from the
stock library into 100 ml of Escherichia coli ER2537 (New England Biolabs. Inc.)
(grown in LB medium with 250 rpm shaking at 37°C to 0060,, approximately 0.4)and
incubated 6.5 hr at 37°C with shaking at 250 rpm. After the culture was centrifuged
at 1,200 x g for 20 min. the supernatant containing amplified recombinant M13
phage was collected and filtered through an Acrodisc Syringe Filter (pore size
0.45pm, low protein binding) (Gelman Science, Ann Arbor, MI). The titer of the
recombinant phage was determined by plaque counting after infection of E. coli
ER2537 with serial 10-fold dilutions of the phage in LB top agar and plating on LB

agar plates.

Phage selection by panning-alution selection

One hundred microliters of mAb 6F5 (15 pglml) in 0.01M phosphate buffered
saline (PBS) (pH 7.4) was dispensed into each well of disposable lmmulon-4
microtiter strips (Dynatech Laboratories, Inc., Chantilly, VA). The antibody was dried
onto the wells in a forced-air oven at 40°C. overnight. Blank wells were coated with
an equal concentration of mouse immunoglobulin G. The wells were washed 4

times by filling each well with 300 pl PBS and aspirating the contents. Nonspecific

binding was blocked by incubating 320 pl of 10% non-fat dry milk dissolved in PBS

128

(10% milk-PBS) in each well for 1 hr at 37°C and then washing 4 times with PBS.

For the panning-alution selection (Figure 5.2), the recombinant phage-display
peptide library (diluted with 10% milk-PBS to approximately 1010 pfulml) was then
added into the walls (100 pllwell) and incubated at 37°C for 1 hr. The wells were
washed 20 times by filling each well with approximately 300 pl PBS containing 0.1%
(vol/vol) Tween 20 (PBS-T) followed by incubation with 300 pllwell of PBS at 37°C
with shaking at 150 rpm for 1 hr. The wells were washed again 10 times with PBS-T
(300 pllwell). To elute the bound phages in the microtiter walls. 100 pl of DON (100
pglml in PBS containing 1% methanol) or PBS containing 1% methanol was added
to each well and incubated at 37°C with shaking at 150 rpm for 1 hr. The eluted
phages were then collected from the microtiter wells and used to reinfect E. coli
ER2537 cells for phage titering and amplification. After four rounds of panning-
elution selection, individual plaques were picked from LB plates and used to infect
2.5 ml of E. coli ER2537 culture described above. After 6 hr incubation at 37°C with
shaking at 250 rpm, the culture was centrifuged at 10.000 x g for 10 min and the
supernatant containing recombinant phages was collected. The binding activity to
mAb 6F5 was tested for each individual phage clone by ELISA. The bound phages
in mAb 6F5 coated microtiter walls was determined by incubation with 100 pllwell
of sheep anti-M13 HRP conjugate (5000 times diluted in 10% non-fat dry milk-PBS)
at 37°C for 1 hr followed by incubation with 100 pllwell of TMB substrate at 37°C
for 15 min. The absorbance was read at 450nm after stopping the reaction with 100

pllwell of 10% H260...

129

 

 

 

 

 

 

 

 

Re-Infectlon and re-ampllficatlon

 

Figure 5.2. Panning-alution selection procedure for deoxynivalenol (DON)
mimotopes in anti-DON monoclonal antibody coated lmmulon-4
microtiter walls.

130

Titration and amplification of panning-elutlon selected phages

A 10-fold dilution series of the selected phages was made in LB medium and
100 pl of the diluted phages was mixed with 100 pl of E. coli ER2537 (grown in LB
with shaking (250 rpm) at 37°C to OD$00 approximately 0.2) and incubated at room
temperature for 30 min for infection. The infected E. coli ER2537 calls were mixed
with 4 ml LB top agar and spread onto LB agar plates. After solidification, the plates
were incubated at 37°C overnight and plaques were counted. To amplify the
phages, approximately 2 ml of phage solution eluted from monoclonal antibody 6F5
coated walls was added into 50 ml E. coli ER 2537 (grown in LB with shaking at 250
rpm at 37°C to 0060,, 0.2) in a 250 ml flask and incubated for 6 hr at 37°C with
shaking (250 rpm). The supernatant from this culture was collected after
centrifugation at 10,000 x g for 10 min. In order to avoid the interference of a high
concentration of free DON in the phage solution. 100 pl of the supernatant
containing amplified phages was added into 50 ml E. coli ER 2537 for re-
amplification as described above. The re-amplified phages, containing
approximately 8 nglml of DON, was collected after centrifugation and used for a

subsequent round of panning-alution selection.

Competitive ELISA with phage-displayed peptlde

For competitive direct ELISA (CD-ELISA) with phage-displayed peptide,
microtiter walls were coated with mAB 6F5 and blocked as described for panning-
alution selection. Various concentrations (0-10.000 nglml in 1% methanol-PBS) of

DON were mixed with an equal volume of phage-displaying peptide (diluted 10

131

times in 10% non-fat dry milk-PBS). The mixtures were added into mAB 6F 5 coated
microtiter wells at 100 pllwell and incubated at 37°C for 1 hr. After washing 6 times
with PBS-T (300 pllwell), the bound recombinant phages were determined by
incubation with 100 pllwell of sheep anti-M13 HRP conjugate (diluted 1:5000 in
10% non-fat dry milk-PBS) at 37°C for 1 hr. Bound enzyme was determined as
described above. For comparison, 50 pllwell of DON-HRP was also mixed with 50
pllwell of DON at various concentrations (0-10.000 nglml in 1% methanol-PBS) and

incubated in mAB 6F5 coated microtiter wells at 37°C for 1 hr. Bound enzyme was

determined as described above.

For competitive indirect ELISA (Cl-ELISA) with phage-displayed peptide, 100
pllwell of phage-displayed peptide was dispensed into each well of disposable
lmmulon-4 microtiter strips and dried onto the wells in a forced-air oven at 40°C
overnight. Plates were washed and blocked as in the CD-ELISA. Various
concentrations (0-10,000 nglml in 1% methanol-PBS) of DON (50 pllwell) were
added into the wells followed by 50 pllwell of anti-DON mAB 6F5 (10 pglml in 10%
non-fat dry milk-PBS). Wells were incubated at 37°C for 1 hr. After washing 6
times with PBS-T, the bound anti-DON mAB 6F5 was determined by incubation
with goat anti-mouse IgG HRP conjugate (diluted 2000 times with 10% non-fat dry

milk-PBS) at 37°C for 1 hr. Bound enzyme was determined as described above.

DNA sequencing
After confirmation of the specific binding to mAB 6F5 by ELISA, 10 ml of

recombinant phage (1011 pfulml in LB) from each positive phage clone was used for

132

single stranded DNA (ssDNA) isolation with QlAprep Spin M13 Kit (QIAGEN Inc.,
Chatsworth, CA). The ssDNA was sequenced with -28 glll sequencing primer and
-96 glll sequencing primer (New England Biolabs. Inc.) by Taq cycle sequencing

and dye terminator chemistry at the Michigan State University Sequencing Facility.

Translational fusion with bacterial alkaline phosphatase (AP)

A 179 bp DNA fragment encoding the 93p signal peptide, DON mimotope
peptide and first 17 amino acids of M13 phage g3p protein was amplified by
polymerase chain reaction (PCR) with Pfu DNA polymerase using a sense primer
(5'-GCCAAGCTTAGATC'I'I'GGAGCCT'ITFITTI'GGAG-3') and an anti-sense
primer (5'-CCGGTCGACCTGTATGGGA‘I'I'T'I’GCTAAACAACT—3'). After gel
purification, the amplified DNA was digested with 89! II and Sal l and cloned into
BamH I-SaII digested leP5 (Carrier at al., 1995) to generate plasmid pOY7. The
ligated product was used to transform E. coliDH11S competent cells (Gibco BRL.
Gaitharsburg. MD), generating DH11S/pQY7. For DONPEP-AP fusion protein
production, DH1IS/pQY7 was grown in SB supplemented with ampicillin (100
pglml) and 2 % glucose at 30°C with shaking (250 rpm) to absorbance (600nm)
(A600) approximately 0.5. After the culture was centrifuged at 1000 x g for 10 min
and the medium was discarded. the cells than were resuspended in SB containing
ampicillin (100 pglml) and 1 mM isopropyI-B-D-thiogalctopyranoside (IPTG) and
incubated with shaking (250 rpm) at 30°C overnight. The supernatant containing the
extracellular soluble DONPEP-AP fusion protein was separated from the cell pellets

by centrifugation at 7.000 x g for 15 min and filtered through a 0.45 pm-pore-size

133

filter. The periplasmic DON PEP-AP fusion protein was extracted by suspending the
bacterial cells (1 :5 vol/vol) in lysis buffer (50 mM Tris-HCI, pH 8.0, 20% sucrose.
10 mM EDTA . 0.1 mg/ml of lysozyma. 0.5 mM PMSF), and left on ice for 1 hr with
agitation before centrifugation for 30 min at 7,000 x g. The supernatant was filtered
through a 0.4 pm porous filter. The activity of DONPEP-AP fusion protein was

determined by CD-ELISA as described below.

CD-ELISA with DONPEP-AP fusion protein

Microtiter wells were coated with mAB 6F5 and washed as described above.
Various concentrations (0-10,000 nglml in 0.05M Tris buffered saline. pH7.4) of
DON were mixed with an equal volume of DONPEP-AP fusion protein (periplasmic
extract diluted 1:7 with 2% non-fat dry milk-TBS). The mixtures (100 pllwell) were
added into each mAB 6F5 coated microtiter well and incubated at 37°C for 1 hr.
After washing 6 times with 300 pl TBS containing 0.1% Tween 20 (TBS-T). the
bound DONPEP-AP was datenninad by incubation with pNPP substrate solution at

37°C for 45 min. The absorbance was read at 405 nm.

CD-ELISA using peptide C430 and its HRP conjugate

One DON mimotope peptide plus a structurally flexible linker and a cysteine
residue, NHZ- SWGPFPFGGGSC-COOH, was synthesized via Fmoc chemistry at
Bio-Synthesis, Inc. (Lewisville, TX) and was named C430. The synthetic peptide
C430, at various concentrations. was used to compete with DON-HRP for binding

to mAB 6F5 in CD-ELISA. C430 was also chemically conjugated to HRP with a

134

sulfo-MBS cross linker. For CD-ELISA with C430-HRP conjugate, the procedure
was the same as CD-ELISA with DON-HRP described above, except that C430-
HRP (diluted 1:5000 in blocking buffer) replaced DON-HRP.

Immunization of animals with C430-BSA conjugate

The synthetic peptide C430 was conjugated to bovine serum albumin (BSA)
with a sulfo-MBS cross linker. The conjugate (approximately 29 : 1 molar ratio of
peptide C430 to carrier protein BSA) was used to inject 7-weaks-old BALB/c female
mice intraperitoneally or 6-month-old New Zaaland white rabbits subcutaneously.
Initial injections for mice were 100-200 pg of C430-BSA conjugate in 200 pl of
saline-Fraund’s complete adjuvant (1 :1 ). followed at 3-weak intervals by boosts of
100-200 pg conjugate in 200 pl saline-Freund’s incomplete adjuvant(1 :1). The initial
injection for rabbits was 1 mg of C430-BSA conjugate in 1 ml of saline-Fraund’s
complete adjuvant (1 :1 ). followed at 4-week intervals by boosts of 250 pg conjugate
in 1 ml saline-Fraund’s incomplete adjuvant (1 :1). Animals were bled 1 week after
each boost. Antisera were screened for specific binding in phage-displayed
DONPEP.2 coated walls by CI-ELISA using C430 or DON as competitive antigens.

Cytotoxicity and effects on protein synthesis in vitro

B6C3F1 mice (10-weeks-old) were euthanized by cervical dislocation and
femurs were removed. Bone marrow cells were flushed from the femurs using a
1 cc syringe and 25 gauge needle. Erythrocytes were Iysed with 0.83% ammonium

chloride. Call number and viability were determined by trypan blue dye exclusion

135

using a hemacytometer. The cells were cultured at 1 X 10" cells/ml in 96 well flat
bottom plates in RPMl-1640 in a 5% CO2 humidified incubator. RPMl-1640 was
supplemented with 100 U/ml penicillin, 100 pglml streptomycin, 50 pM 2-
mercaptoethanol, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM
glutamine and 10% fetal bovine serum. DON and synthetic peptide C430 were
diluted in RPMI-1640. Duplicate cultures were treated. After 18 hr of exposure to
DON and synthetic peptide C430, cell viability was determined using an MTT
conversion assay as described by Marin et al. (1996).

To determine the effects on protein synthesis, DON or synthetic peptide
C430 (0 or 3.4 pM in the final 50 pl reaction) was added to 30 pl biotin translation
mix (Boehringer Mannheim Corporation, Indianapolis, IN) containing reticulocyte
lysate, 10 pmol biotin-lysine-tRNA'”, 42 pM amino acids without lysine, sperrnidine,
energy mix, dithiothreitol, 83 mM K-acetate, and 0.83 mM Mg-acetate. The mixtures
(46 pl) were incubated on ice for 10 min, followed by the addition of 4 pl of y-globulin
mRNA (0.5 pg/pl). The final reaction (50 pl) was incubated at 30°C for 1 hr. The
translated protein samples were stored at -80°C.

For Western blot analysis, 3 pl of protein translated in vitro were boiled for
10 min after mixing with SDS-PAGE loading buffer. The samples then were loaded
into a mini SDS-10% polyacrylamide gel and subjected to electrophoresis at 80 V
for 2 hr. The newly synthesized proteins were detected by electrotransfer to a
PVDF transfer membrane (DuPont NEN Research Products, Boston, MA), followed
by incubation with streptavidin-peroxidase conjugate (Boehringer Mannheim

Corporation). Bound enzyme was visualized by incubation with SuperSignal"

136

ULTRA chemiluminescent substrate (Pierce Chemical Company) and exposing a

Kodak XAR5 autoradiography film (Kodak, Rochester, NY).

137

RESULTS

Panning-alution selection of specific phages

After each round of panning-elution selection, the numbers of eluted phages
were monitored (Figure 5.3). Enrichment for the specific phages with affinity to mAb
6F5 was observed after two rounds of panning-elution selection. The phages with
affinity to mAb 6F5 were enriched approximately six orders of magnitude after the
fourth round of panning-elution. Fifteen individual phage plaques from the fourth
round of panning-elution selected phages were randomly isolated to infect E. coli
ER2537 for phage reamplification. Each of the 15 phage isolates was tested for
their binding activity to mAb 6F5 by ELISA. Five of these phage isolates, named as
DONPEP.1, DONPEP.2, DONPEP.3, DONPEP.4, and DONPEP.12, showed
binding activity to mAb 6F5.

Single stranded phage DNA was isolated and the nucleotide sequences were
determined for each of the five positive phage isolates. Four of five sequenced
isolates had identical DNA sequence (5'-AG'|‘|'GGGGTCCTITTCCG'|'TT—3')
encoding the consent peptide sequence SWGPFPF, while isolate DONPEP.12
differed by 4 nucleotides (5'-1§TTGGGGTCC§_QTTCCTTTT-3') resulting in a

change of the fifth amino acid from F5 to L5.

138

 

 

 

 

 

 

h4011513186 coated

[:1 mAb 6F5 coated

 

 

 

 

 

           
    

 

101° —

0

06

04
102 —
10°

1.55.3 6820 owns—m

2nd 3rd 4th

lst

Round of biopanning selection

microtiter wells or from mouse IgG coated lmmulon-4 microtiter wells
139

elution with DON (100 pglml) from mAb 6F5 coated lmmulon-4
in each round of panning-elution selection.

Figure 5.3. Enrichment of mAb 6F5 specific phage. Phage were titered after

Application of DON mimotope peptide in competitive ELISA

To demonstrate whether the positive recombinant phage peptides actually
mimic the epitope recognized by mAB 6F5 or just bind non-specifically to the
surface of the antibody molecule outside the antigen binding site, two different
positive phage clones were tested for binding to mAB 6F5 in a CD-ELISA (Figure
5.4). Binding of phage clone DONPEP.2 and DONPEP.12 to immobilized mAB 6F5
was competitively inhibited by free DON. This strongly suggested that these two
phage clones were binding to the antigen binding site of the monoclonal antibody,
mimicking, in part, the structural epitope of DON.

To confirm that the sequence obtained from the phage library binds
specifically to mAB 6F 5 independently of phage structural context, synthetic peptide
C430 was tested for binding in CD-ELISA. Binding of DON-HRP to immobilized
mAB 6F5 was inhibited by free C430 (Figure 5.5A) while the binding of C430-HRP
conjugate to immobilized mAB 6F5 was conversely inhibited by free DON (Figure
5.58). This suggested that the peptide was by itself sufficient for binding to the
antibody, independent of the phage structural context. When C430 was used to
compete DON-HRP or C430-HRP for binding to mAB 6F5, the leo's were 0.64 to
0.8 pM, whereas 3.4 pM of free DON was required to reach 50% inhibition of DON-
HRP or C430-H RP binding to the same antibody (Table 5.1). This indicates a higher
affinity of mAB 6F5 for C430 than for DON. in a similar CD-ELISA, none of the
individual amino acids (at concentrations up to 34 pM) in C430 showed any

significant binding inhibition to DON-HRP (Figure 5.6). This suggests that the

140

Figure 5.4.

Absorbance at 450 nm

 

0.8 -~

0.6 4-

   

 

 

 

0-4 ‘“ +00»an
+Phagc DONPEP.2
0,2 __ +Phage DONPEP.12
0 l i ‘r
0 10 100 1000 10000

Competitive DON, ng/ml

Competition between phage-displayed mimotope peptides and DON
for binding to immobilized mAb 6F5 in CD-ELISA. Various
concentrations of free DON competed with an equal volume of phage-
displayed peptides (at constant concentration) for binding to
immobilized mAb 6F5. Bound phage peptide was detected by
horseradish peroxidase conjugated sheep anti-M13 lgG and then
measured by absorbance. DON-HRP was included as a positive
control.

141

 

 

   
 
 
 
 
 
 
 

  
   
 
 

 

 

 

 

100 100
°\o —D—DON °\° 90 - -D-DON
g3 30‘ +C430 g3 80‘ +080
=5 'e 70 ‘
f5 60 - g 60 -
“5 “a 50 ‘
g 40 - g 40 ~
:g :e 30 -
E 20 « E 20 .
10 -
o 0 ~
0 0.01 0.1 1 10 100 o 0,01 0,1 1 10 100
Competitor, PM Competitor, pM

Figure 5.5. Competition between synthetic peptide C430 and DON for binding to
mAb 6F5. (A). DON and synthetic peptide C430 compete with DON-
HRP for binding to immobilized mAb 6F5. (B). DON and synthetic
peptide C430 compete with C430-H RP for binding to immobilized anti-
DON mAb 6F5.

142

Table 5.1.

50% binding inhibition of DON-HRP and C430-HRP by DON and
C430 in competitive ELISA with monoclonal antibody 6F5 coated in
microtiter wells.

 

 

 

 

 

Enzyme Binding competitor ( pM)
conjugate DON C430
DON-HRP 3.43 0.64
C430-HRP 3.47 0.8

 

 

 

 

Absorbance at 450 nm

Figure 5.6.

 

 

 

 

 

 

 

 

 

0'8 +Ser (S) +Trp (W)

+Gly (G) -o—Pro (P)

0.4 ~ —o—Phe (F) +Cys (C)

0 . T r .
0.001 0.01 0.1 1 10 100

Competitive amino acid, pM

Competition between individual amino acids in synthetic peptide C430
and DON measured with CD-ELISA. Amino acids (at various
concentrations) competed with DON-HRP for binding to immobilized

mAb 6F5.

143

100

 

  
   
  
  
  
  
  
  
 
    

9O -
+DONPEP-AP
8O -

-D—DON-HRP
7o —
60 J
50 -

40~

Inhibition of binding, %

30‘
20~
10-

 

 

1

0.001 0.01 0.1 1 10 100

Competitive DON, pM

l l l
l T l rrrrrr t It IT

Figure 5.7. CD-ELISA with DONPEP-AP fusion protein. Binding of DONPEP-AP
fusion protein to immobilized mAb 6F5 was inhibited by free DON.
DON-HRP was included as a positive control.

144

sequence in C430 is important for the specific binding to mAB 6F5, while the
individual amino acids can not bind to mAB 6F5 specifically.

The DONPEP-AP fusion protein collected from the culture supernatant or
periplasmic extract showed alkaline phosphatase activity and had a similar specific
binding to mAB 6F5 as DON-HRP (Figure 5.7). This suggests that the DON
mimotope peptide sequence is structurally stable in a different protein structural

context.

CD-ELISA for DON spiked wheat extract

To test the feasibility of DON mimotope peptide sequences as
immunochemical reagents for a DON immunoassay in food and feed, a CD-ELISA
was performed for a DON spiked wheat extracts. Both C430-HRP conjugate and
DONPEP-AP fusion protein showed binding activity to immobilized mAB 6F5 in the
wheat extract (Figure 5.8). All three conjugates had a similar linear inhibition curve
at DON concentrations ranging from 0.1 pglml to 10 pglml in the wheat extract.
However, a slightly lower absorbance was observed in the CD-ELISA with C430-
HRP and DONPEP-AP in wheat extract than in PBS buffer, indicating that the
wheat extract interfered with the binding of the mimotope peptide to anti-DON mAB

6F5 to some degree.

145

Inhibition of binding, %

Figure 5.8.

100

 

 
  
    
   

(D
O

-D- DON-HRP
-I- DONPEP-HRP

 

 

0 10 100 1000 10000
Spiked DON (nglml)
Application of C430-HRP and DONPEP-AP in DON immunoassay
(CD-ELISA) in a DON-spiked wheat extract. Immulon-4 microtiter

wells were coated with mAb 6F5 antibody. DON-HRP was included
as a positive control.

146

Immunization with C430-BSA conjugate

If the selected peptides represent an true image of the DON surface
structure, they might be able to elicit an immune response similar to that of the
original DON epitope, and therefore they might serve as alternative antigens for
DON. When rabbits and mice were immunized with C430-BSA conjugate, both
produced antibody specific to the DON mimotope peptide sequence after the
second injection of C430-BSA. After four injections, the antiserum showed a strong
antibody response against the DON mimotope peptide sequence (Figure 5.9). A
concentration of 0.39 pM of synthetic peptide C430 caused 50% inhibition of
antiserum binding to immobilized phage-displayed peptide. However, binding of
antisera to immobilized phage-displayed peptide was not inhibited by free DON in

solution, indicating that antibodies in these immunized animals were not specific for

DON.

Cytotoxicity and effects on protein synthesis in vitro
One of DON’s effects is to cause cytotoxicity through cell apoptosis (Pestka
et al., 1994b). To test if the DON mimotope peptide is cytotoxic, synthetic peptide

C430 was compared with DON for their cytotoxic effects to bone marrow cells. As
expected, DON at 3.4 pM caused 40-60% cell death after 18 hr incubation (Figure
5.10). Synthetic peptide C430, however, did not show any adverse effects on the
viability of bone marrow cells in culture at concentrations up to 34 pM (Figure 5.10).

When combined with DON, C430 at any of the tested concentrations did not

147

Figure 5.9.

 

Absorbance at 450 nm

 

 

O .......l “he... ..... ..... ........
0.001 0.01 0.1 1 10 100

 

 

Competitor, pM

The specificity of antibody from C430-BSA immunized mice. DON and
C430 at various concentrations were used to inhibit the binding of
antisera to immobilized DONPEP.2 phage. Bound mouse antibodies

were detected by horseradish peroxidase conjugated goat anti-mouse
lgG and measure by absorbance.

148

 

 

34

 

 

 

 

120

 

x 5:52., :8

Synthetic peptide C430, pM

peptide C430 co-incubated with (white bar) or without (hatched bar)
DON (3.4 pM). Cell viability was determined using a MTT conversion

Figure 5.10. Cytotoxicity to bone marrow cells after 18 hr incubation with synthetic
assay.

149

significantly increase or decrease cell viability caused by DON, suggesting that no
significant synergism or antagonism between DON mimotope peptide and DON in
causing bone marrow cell death.

In a cell free translation system, at a concentration 3.4 pM, DON significantly
inhibited new protein synthesis, while C430 showed no inhibition (Figure 5.11).
However, protein synthesis was not inhibited by DON when mixed with C430 both
at 3.4 pM. This suggests that C430 was antagonistic to the inhibitory effects on

protein synthesis caused by DON.

150

 

 

 

Figure 5.11. Effects of DON (3.4 pM) and synthetic peptide C430 (3.4 pM) on
protein synthesis in vitro with rabbit reticulocyte lysate. The translation

template was y-globulin mRNA (2 pg). “+" or ‘-": with or without
added, respectively.

151

DISCUSSION

In a search for DON mimotope peptides, we screened a phage-display
random- heptapeptide library and identified two closely related peptide sequences
that specifically bind with high affinity to anti-DON mAb 6F5. To our knowledge, this
is the first example of a mimotope peptide for a nonproteinaceous, low-molecular-
weight mycotoxin.

In a competitive ELISA, the binding of the selected peptides to mAb 6F5
could be competed by DON, while DON binding to the same antibody was also
competed by the synthetic peptide, strongly suggesting that these clones bind to the
same antigen-binding site of the monoclonal antibody. The demonstration that these
peptides can be applied to ELISA has several implications. From a safety
standpoint, it is advantageous to use a nontoxic peptide as an immunochemical
reagent to replace toxic DON in the immunoassay. Second, the conjugation of
peptide to peroxidase or other proteins will be much easier than the conjugation of
DON to the same enzyme. Finally, by genetically fusing the peptide with reporter
enzyme such as alkaline phosphatase, chemical conjugation can be avoided.

Although many mimotope peptides for protein antigens have been isolated
from phage-displayed peptide libraries, only a few examples exist where peptides
were selected as mimics to non-proteinaceous chemicals, such as lectins and
carbohydrates (Oldenburg et al., 1992; Scott et al., 1992; Hoess et al., 1993). It is
interesting to compare the peptide sequences selected with mAb 6F5 to other

peptide sequences known to interact with carbohydrate binding sites. One can ask

152

whether there are general features common to these sequences. Our sequences
are similar to those carbohydrate mimics in that there is a clear preference for
aromatic groups and proline residues (Oldenburg et al., 1992; Scott et al., 1992;
Hoess et al., 1993). This is not unreasonable since these amino acids resemble
sugar moieties in their size and cyclic shape. Furthermore, the proline may serve
an important structural role by orienting the aromatic side chains spatially in a
manner similar to the branched carbohydrates they mimic.

if a small peptide completely mimics the epitope of a certain antigen, it can
elicit antibodies directed against native antigen whose epitope the peptide
represents after the peptide is conjugated on a suitable carrier protein or displayed
on phage (Amon, 1991; Minenkova et al., 1993; Di-Marzo et al., 1994; Motti et al.,
1994). But peptides mimicking discontinuous epitopes were unable to elicit
antibodies specific to the original antigen (Felici et al., 1993). Although our selected
peptides and DON competed with each other for binding to mAB 6F5 and C430 can
antagonize DON’s inhibitory effects in an in vitro protein synthesis system, no
specific antibodies for DON were produced from C430-BSA conjugate immunized
animals when high affinity antibodies to the peptide were detected in these animals.
It is not clear whether the selected peptides mimic the surface structure of DON in
some respects or rather bind to the cleft by an entirely different mechanism (Figure
5.12). The latter possibility has been noted in the case of streptavidin-binding
peptides discovered with a phage-display library (Weber et al., 1992), where the
peptides associate with the binding site in a quite different fashion than the natural

ligand, biotin. Turcatti et al. (1997) also observed that non-peptide antagonist and

153

Model 1 Model 2

Binding to mAb 6F5 Binding to mAb 6F5

 

   

 

 

Generation of DONPEP antibody Generation of DONPEP antibody
after immunized with DON PEP after immunized with DONPEP

 

   

Figure 5.12. Possible mechanisms for the binding of DON mimotope peptides to
mAb 6F5 and for the immunogenicity of C430-BSA. In Model 1, DON
and DONPEP might bind to different portions at the antigen binding
pocket of mAb 6F5. After immunization with DONPEP, only anti-
DONPEP antibody is produced. In Model 2, DON and DONPEP bind
to mAb 6F5 in the same fashion. But after immunization with
DONPEP, only the epitope which is different from DON elicits
antibody response.

154

peptide agonists were vastly different when bound to the NK1 receptor. Another
possibility is that more than one epitope may exist in the peptide and the moiety
which mimics DON has very poor immunogenicity (Figure 5.12). This proposed
mechanism is supported by the observation that specific antibody for DON was
rarely detected in large numbers of animals immunized with DON carrier protein
conjugates (Casale etal., 1988). The toxic effects of trichothecenes are
presumably related to their protein synthesis inhibition. The inhibitory activity of
trichothecenes is due to their ability to bind the 608 subunit of eukaryotic ribosomes
and to interfere with peptidyltransferase activity (Bamburg, 1983; Ueno, 1983;
Thompson and Wannemacher, 1986; Betina, 1989b; Rotter et al., 1996).
Stoichiometricaly, one trichothecene molecule binds one ribosome (Middlebrook
and Leatherrnan, 1989). Presumably peptides will only be agonists if they undergo
the same specific contacts and interactions with the ribosome as DON. The
mimotope peptides selected from phage-display library in this study, however,
showed an antagonism to DON-induced protein synthesis inhibition. This suggests
that the synthetic peptide may bind to the same site in ribosome as DON does, but
in a different fashion. It is commonly accepted that native ligands, agonists, or
antagonists can partly bind to their receptors (Strader et al., 1995) although an
inhibitory active site might be structurally distinct from the binding site. it is notable,
however, that in a cell culture model, C430 did not potentiate or attenuate DON in
causing cell death. This may be due to the difficulty of the peptide crossing the cell

membrane (Burton et al., 1996; Firth et al., 1997). DON, with a 4-fold lower

155

molecular weight, absence of charged groups, and specific structure, may be more
readily absorbed into the cell (Witt and Pestka, 1990).

In addition to the toxic effects to animals and humans, there is a growing
body of evidence indicating that trichothothecenes, including DON, can enhance the
virulence of plant-pathogenic species of Fusarium on plant hosts (Adams and Hart,
1989; Desjardins et al., 1989:1992; Bmins et al., 1993; Wang and Miller, 1988;
Scholbrock et al., 1992; Atanassov et al., 1994; Proctor et al., 1995). These
trichothecene-producing Fusarium fungi cause a broad range of plant diseases
including head and seedling blight of small grains such as wheat and rye, ear and
stalk rot of maize, stem rot of carnation, and seedling blight and root rot of a number
of other plant species, including beans, clover, and tomato (Cook, 1981;
Kommedahl and Windels, 1981). If the fungal virulence associated with DON is
mediated through protein synthesis inhibition and the mimotope peptides presented
here are antagonistic to DON activity in plant, a transgenic plant expressing the
peptides (may need to be fused into another protein) might show reduced levels of
wheat head scab and other plant diseases caused by DON-producing pathogenic
Fusarium. Previously, synthetic peptide combinatorial libraries have been used to
identify bioactive peptides against plant pathogens (Marcos et al., 1995; Blondelle
and Houghten, 1996; Reed et al., 1997). Some antimicrobial peptides have been
expressed in transgenic plants (De Bolle et al., 1996). Thus, phage-display peptide
libraries may provide a new and innovative approach in developing antimicrobial

peptides to control plant diseases.

156

In summary, we have identified two closely related mimotope peptides for
DON and demonstrated their potential application in DON immunoassay. We also
demonstrated an antagonistic activity of the mimotopes to DON-induced protein
synthesis inhibition. Further understanding of the difference between the mimotope
peptides and DON in the structure-function relationships might help in elucidation
of the mechanisms of DON toxicity, and lead to prevention of plant and animal

diseases associated with this mycotoxin.

157

SUMMARY

In this dissertation, phage—display technology was explored to develop
recombinant antibodies and peptide mimotopes for Fusarium mycotoxins. Firstly,
a functional scFv antibody with high affinity to the mycotoxin zearalenone was
cloned and expressed in E. coli. A new phage-display vector which facilitates the
cloning of genes for variable heavy chain, variable kappa light chain, and the
assembly of scFv DNA fragment, was also constructed. However, attempts to
generate scFv antibodies to the mycotoxin fumonisin B1 from fumonisin B1-cholera
toxin conjugate immunized mice failed due to inefficient selection. Secondly, the
anti-zearalenone scFv DNA fragment was transferred into Arabidopsis and
functional anti-zearalenone scFv plantibody with high affinity to mycotoxin
zearalenone was expressed. Finally, two highly homologous heptapeptide
sequences from a phage-displayed random 7-mer peptide library were identified for
specifically binding a deoxynivalenol-specific monoclonal antibody. The mimotope
peptide appeared to be nontoxic and showed antagonistic to mycotoxin

deoxynivalenol.

158

APPENDICES:
BUFFERS AND MEDIA

159

APPENDIX A: COMMONLY USED BUFFERS

Bacteria alkaline lysis buffer for mini-preparations of plasmid DNA
(Sambrook et al., 1989):

Solution I
50 mM glucose
25 mM Tris.Cl (pH 8.0)
10 mM EDTA (pH 8.0)
Autoclaved and stored at 4°C

Solution ll (fresh prepared)
0.2 N NaOH (freshly diluted from a 10 N stock)

1% SDS

Solution ill
5 M potassium acetate 60 ml
glacial acetic acid 11.5 ml
H20 28.5 ml

0.05 M carbonate-bicarbonate coating buffer (Sigma Chemical Company,
St. Louis, MO):

1.59 g Na.‘,CO3

2.93 g NaHCOa

dissolved in 1 liter Milli-Q water

adjust pH to 9.6, if necessary

stored at 4°C

Citrate buffer (pH 4.0) for ABTS substrate preparation:
Add 9.6 g of citric acid monohydrate to 500 ml of Milli-Q water. Adjust the pH
to 4.0 with NaOH. Scale up to 1 liter and check pH. Autoclave and store at
4°C.

DNA electrophoresis gel-loading buffer (Sambrook et al., 1989):

Buffer type i (6 x)
0.25% bromophenol blue
0.25%xylene cyanol FF
40% (wlv) sucrose in water
4°C storage

160

Buffer type II (6 x)
0.25% bromophenol blue
0.25%xylene cyanol FF
15% Ficoll (Type 400; Pharmacia) in water
room temperature storage

Buffer type ill (6 x)
0.25% bromophenol blue
0.25%xylene cyanol FF
30% glycerol in water
4°C storage

Buffer type iv (6 x)
0.25% bromophenol blue
40% (wlv) sucrose in water
4°C storage

Phosphate-buffered saline (PBS) (Sambrook et al., 1989):

To 8 g of NaCl, 0.2 g of KCI, 1.44 g of Na,HPO,, 0.24 g of KHZPO4, add Milli-
Q water to a total volume of 1 liter. Adjust the pH to 7.4 with NaOH or HCI if

necessary and autoclave.

0.05 M phosphate-citrate buffer for TMB substrate preparation (Sigma

Chemical Company, St. Louis, MO):
Dissolve 3.65 g of NazHPO, and 2.55 g of citric acid in Milli-Q water, scale
up the volume to 500 ml. Adjust pH to 5.0, if necessary.

Protein SDS-PAGE gel-loading buffer (2 x) (Sambrook et al., 1989)

100 mM Tris.CI (pH 6.8)

200 mM dithiothreitol (add just before the buffer is used)

4% SDS (electrophoresis grade)

0.2% bromophenol blue

20% glycerol

room temperature storage (without dithiothreitol)

SDS-PAGE running buffer (5 x stock) (Sambrook et al., 1989):

15.1 g Tris base

94 g glycine

5 9 SDS (electrophoresis-grade)
add Milli-Q water to 1 liter

161

SDS-PAGE protein transfer buffer
12.1 g Tris base
14.4 g glycine
add Milli-Q water to 1 liter

Tris-buffered saline (TBS) (Boehringer Mannheim Corporation,
Indianapolis, IN ):

Add 6.05 g Tris base (50mM), 8.76 g NaCI (150 mM) to 800 ml Milli-Q water,

adjust pH to 7.5 with HCI or NaOH, scale up to a total volume of 1 liter with
Milli-Q water.

Tris-EDTA (TE) buffer (Sambrook et al., 1989):

pH 7.4:

10mM Tris.Cl (pH7.4)
1mM EDTA (pH8.0)

pH 7.6:

10mM Tris.Cl (pH7.6)
1mM EDTA (pH8.0)

pH 8.0:
10mM Tris.Cl (pH8.0)
1mM EDTA (pH8.0)

Tris-acetate electrophoresis buffer (T AE, 50 x stock) (Sambrook et al.,
1989y

242 g Tris base

57.1 ml glacial acetic acid

100 ml 0.5 M EDTA (pH 8.0)

scale up to 1 liter with Milli-Q water

162

APPENDIX B: COMMONLY USED MEDIA

LB medium (Luria-Bertani medium) (Sambrook et al., 1989)
10 g bacto-tryptone
5 g bacto-yeast extract
10 9 NaCl
dissolved in Milli-Q water, adjust pH to 7.4
scale up to 1 liter, autoclave.

Minimal medium (Phannacia blotech):
6 g NazHPO, (dibasic)
3 g KH,,PO4
1 g NH,C|
Dissolved in Milli-Q water (500 ml), adjust pH to 7.4, autoclave;

In a separate bottle, autoclave 15 g of Bacto-agar in 492 ml Milli-Q water,
autoclave. Add 1 ml of1 M MgCl2 .6H20, 1 mi of 1 M CaCl2.2H20, 1 ml of 1
M thiamine hydrochloride and 5 ml of 20% glucose (all autoclave or filtration
sterilized). Then combine with above salts.

SB medium (Phannacia blotech):
35 g bacto-tryptone
20 g bacto-yeast extract
5 9 NaCl
Dissolved in Milli-Q water, scale up to 1 liter, adjust pH to 7.4, autoclave.

SOB medium (Sambrook et al., 1989):
20 g bacto-tryptone
5 g bacto-yeast extract
0.5 9 NaCl
10 ml 250 mM KCI
Dissolved in Milli—Q water, adjust pH to 7.4, scaled up to 990 ml with Milli-Q
water. Autoclave, after cool down to 60°C, add 10 ml of 1 M MgCl,.

2 X YT medium (Sambrook et al., 1989):
16 g bacto-tryptone
10 g bacto-yeast extract
5 9 NaCl
Dissolved in Milli-Q water, adjust pH to 7.4, scale up to 1 liter, autoclave.

163

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