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1999 Development of Immunochemical Methods for Gossypol Analysis. Xi Wang Louisiana State University and Agricultural & Mechanical College
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEVELOPMENT OF IMMUNOCHEMICAL METHODS FOR GOSSYPOL ANALYSIS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Food Science
by Xi Wang B.S., Wuxi Institute of Light Industry, China, 1991 M.S., Wuxi Institute of Light Industry, China, 1994 August, 1999
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9945748
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS
The author wishes to express her sincere appreciation to her present advisor, Dr.
Leslie C. Plhak, Department of Food Science, Louisiana State University, for her
guidance, assistance, and inspiration during this study.
Sincere thanks are given to Dr. Peter J. Wan (USDA, New Orleans, LA), Dr.
Douglas L. Park (Professor and Head of Department of Food Science, LSU), Dr.
Witoon Prinyawiwatkul (Department of Food Science, LSU) and Dr. William
Wischusen (Department of Biological Science) for serving on the dissertation
committee and for their valuable advice.
Grateful appreciation is expressed to Dr. Michael K. Dowd (USDA, New
Orleans, LA), Dr. Millard C. Calhoun (Texas A&M, College Station), Dr. Maren
Hegsted (Department of Human Ecology, LSU), and Laurie A. Henderson (Animal
Care Facility, LSU) for their valuable assistance and suggestions. Special thanks are
given to research colleagues, Eun-Sun Park and Chih-Chun Huang, for their friendship
and assistance.
Sincere appreciation is given to Cotton Incorporated (Raleigh, NC) for financial
support.
Finally, the author would like to express her gratitude to her husband, Feng
Chen, her daughter, Rachel W. Chen, her parents and her sisters for their love, moral
support and encouragement.
ii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
ACKNOWLEDGMENTS------ii
LIST OF TABLES------v
LIST OF FIGURES------vi
LIST OF ABBREVIATIONS------viii
ABSTRACT ------xi
CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE------1 1.1: Overview of cotton and cottonseed products ------1 1.2: Gossypol ------3 1.2.1: Occurrence of gossypol ------3 1.2.2: Physiochemical properties of gossypol ------5 1.2.3: Utilization and physiological properties of gossypol ------12 1.2.4: Toxicity and detoxification of cottonseed products ------14 1.2.5: Gossypol analysis ------19 1.3: Theory of enzyme immunoassay ------22 1.3.1: Enzyme immunoassay ------22 1.3.2: Antibody structure ------24 1.3.3: Antibody-antigen (Ab-Ag) interactions ------26 1.3.4: Polyclonal antibody (PAb) and monoclonal antibody (MAb) production ------26 1.4: Research objectives ------32
CHAPTER2 MATERIALS AND METHODS------34 2.1: Materials------34 2.2: Methods ------36 2.2.1: Instrumentation ------36 2.2.2: Modeling and statistical analysis ------37 2.2.3: Preparation of experimental solutions ------38 2.2.4: Preparation of immunogen ------39 2.2.5: Preparation of solid-phase conjugates ------41 2.2.6: Measurement of gossypol groups in conjugates ------41 2.2.7: Polyclonal antibody (PAb) production ------42 2.2.7.1: Rabbit immunization ------42 2.2.7.2: Antibody capture noncompetitive ELISA for screening sera-- 42 2.2.7.3: Optimization of ELISA ------43 2.2.8: Monoclonal antibody (MAb) production ------44 2.2.8.1: Immunization of mice and serum test ------44 2.2.8.2: Myeloma cell culture ------45
iii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.8.3: Recovery of splenocytes ------46 2.2.8.4: Production of hybridomas ------46 2.2.8.5: Culture, screening, and selection of hybridomas ------46 2.2.8.6: Cell cloning ------47 2.2.8.7: In vitro production of monoclonal antibodies (MAb) ------49 2.2.8.8: Purification of monoclonal antibodies (MAb) ------49 2.2.8.9: Freezing, storage and thawing of cell lines ------49 2.2.9: Crystallization of isomeric gossypol-acetic acid ------50 2.2.10: Preparation of various forms of gossypol ------50 2.2.11: Antibody capture competitive ELISA ------51 2.2.12: Gossypol analysis in cottonseed products ------52
CHAPTER 3 RESULTS AND DISCUSSION------54 3.1: Effects of reducing agent and pH on production of protein-gossypol conjugates—54 3.2: Production of polyclonal antibodies (PAb) ------59 3.2.1: Monitoring PAb production ------59 3.2.2: Optimization of ELISA for PA b ------59 3.2.2.1: Effects of microtiter plates on immobilization of BSA-G 59 32.2.2: Selection of assay buffers ------62 3.2.2.3: Effects of incubation time and temperature on interactions of Ab-Ag ------69 3.2.2.4: Effects of solvents on antibody capture noncompetitive ELISA------72 3.2.2.5: Effects of Ab and BSA-G concentrations ------75 3.2.3: Effects of immunogen preparation method on PAb production 75 3.3: Production of monoclonal antibodies ------78 3.3.1: Mouse immunization ------78 3.3.2: Production and selection of hybridomas ------80 3.3.3: Correlation between cell growth and antibody production ------81 3.4: Effects of reaction pH and structure of derivatizing reagents on gossypol derivatization ------82 3.5: Isolation of (+/-)-gossypol isomers ------86 3.6: Evaluation of antibody specificity using different forms of gossypol in an antibody capture competitive ELISA ------86 3.7: Application of ELISA for the analysis of gossypol in cottonseed products ------100 3.8: Future research ------107
CHAPTER4 SUMMARY AND CONCLUSIONS------109
REFERENCES------111
APPENDICES------123
VITA ------129
iv
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES
1. Cottonseed production and main producers ------1
2. Gossypol groups per BSA in BSA-G conjugates ------55
3. Competition using gossypol-lysine as competitor ------80
4. Detection limit for antibodies using gossypol derivatives as competitors ------97
5. Gossypol contents of cottonseed products ------101
v
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES
1. Chemical structure of gossypol ------4
2. Tautomers of gossypol ------7
3. Antibody capture competitive ELISA ------23
4. Antibody basic unit structure ------25
5. Diagrammatic representation of the production of PAb (polyclonal antibodies) from rabbits------28
6. Diagrammatic representation of the production of MAb (monoclonal antibodies) from m ice ------30
7. The HAT selection system ------31
8. Gossypol-protein conjugate formation via a Schiff base intermediate ------40
9. MALDI-TOF-MS spectrograms of BSA and BSA-G conjugates ------56
10. MALDI-TOF-MS spectrograms of BSA and BSA-G conjugates ------57
11. MALDI-TOF-MS spectrograms of BSA and BSA-G conjugates ------58
12. Antibody production during immunization period from rabbit #16 ------60
13. Antibody production during immunization period from rabbit #15 ------61
14. Comparison of microtiter plates on ELISA for rabbit #16 PAb ------63
15. Comparison of microtiter plates on ELISA for rabbit #15 PAb ------64
16. Comparison of blockers on ELISA for rabbit #15 PAb ------66
17. Effect of dilution buffer on ELISA for rabbit #16 PAb ------67
18. Effect of dilution buffer on ELISA for rabbit #15 PAb ------68
19. Effect of incubation time and temperature on Ab-Ag interactions for rabbit #16 PAb------70
20. Effect of incubation time and temperature on Ab-Ag interactions for rabbit #15 PAb------71
vi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21. Effect of acetone concentration on ACN-ELISA ------73
22. Effect of N,N-dimethylformamide concentration on ACN-ELISA ------74
23. ELISA checkerboard for rabbit #16 PAb ------76
24. ELISA checkerboard for rabbit #15 PA b ------77
25. Antibody production from mice after four immunizations ------79
26. The growth rate of MAb cell line ------83
27. MAb production during culture ------84
28. Absorption spectra of gossypol and gossypol derivatives in 10% methanol ------85
29. HPLC chromatograms of (+/-)-gossypol isomers ------87
30. ELISA inhibition curves using gossypol and different gossypol derivatives for rabbit #15 PAb------89
31. ELISA inhibition curves using additional gossypol derivatives for rabbit #15 PAb------90
32. ELISA inhibition curves using different gossypol derivatives for MAb ------91
33. ELISA inhibition curves using different gossypol derivatives for MAb ------92
34. ELISA inhibition curves using different gossypol derivatives for MAb ------93
35. Effects of gossypol storage time on ELISA inhibition curves for rabbit #15 PAb - 95
36. Effects of gossypol storage time on ELISA inhibition curves for MAb ------96
37. Comparison of (+/-)-gossypol derivatives in ACC-ELISA for MAb ------98
38. Comparison of (+/-)-gossypol derivatives in ACC-ELISA for MAb ------99
39. Total Gossypol in cottonseed samples analyzed by ELISA (10% methanol for sample delivery) and AOCS official method ------102
40. Free Gossypol in cottonseed samples measured using ELISA (10% methanol for sample delivery) and AOCS official method ------104
41. Free Gossypol in cottonseed samples determined using ELISA (10% acetone for sample delivery) and AOCS official method ------106
vii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF ABBREVIATIONS
A/Ao relative absorbance (see Section 2.2.2)
Ab antibody
Abs. absorbance
ABTS 2,2’-azino-bis (3-ethylbenthiazoline-6-sulfonic acid)
ACC-ELISA antibody capture competitive ELISA
Ag antigen
AOCS American Oil Chemists’ Society
ATP adenosine triphosphate
BG Bound Gossypol
BSA bovine serum albumin
BSA-G bovine serum albumin-gossypol conjugate
ca about
ACN-ELISA antibody capture noncompetitive ELISA
CSM cottonseed meal
CV coefficient of variation
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DNFB dinitrofluorbenzine
El enzyme immunoassay
EI-MS electron impact-MS
ELISA enzyme linked immunosorbent assay
viii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FDA Food and Drug Administration
FG Free Gossypol
FIA fluoroimmunoassay
g gram
GLC gas-liquid chromatography
HAT hypoxanthine, aminopterin and thymidine
HB high binding
HBX high binding extra
HGPRT hypoxanthine guanine phosphoribosyle transferase
HIV human immunodeficiency virus
HPLC high performance liquid chromatography
h hour(s)
I50 concentration of analyte giving 50% reduction in absorbance
Ig immunoglobulin (can be IgM, IgG, etc.)
IR infrared spectroscopy
K.eq equilibrium constant
LPH Limulus polyphemus hemolymph
LPH-G Limulus polyphemus hemolymph-gossypol conjugate
MAb monoclonal antibodies
MALDI-TOF matrix-assisted laser desorption ionization-time of flight
min minute(s)
MS mass spectrometry
MW molecular weight
ix
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MWCO molecular weight cut off
nm nanometer
NMR nuclear magnetic resonance
NZW New Zealand White
°C degree Celsius
PAb polyclonal antibodies
PBS phosphate-buffered saline
PBST phosphate-buffered saline with Tween 20
PEG polyethylene glycol
PVC polyvinylchloride
RIA radioimmunoassay
RPMI 1640 Rosewell Park Memorial Institute 1640
RT room temperature
sc. subcutaneous
TG Total Gossypol
TLC thin layer chromatography
TNBS trinitrobenzinesulfonic acid
Tween 20 polyoxyethylene-20-sorbitanmonolaurate
UV ultraviolet
pi microliter
pM micromolarity
pm micrometer
x
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT
Gossypol is a reactive polyphenolic compound present in the cotton plant.
During cottonseed processing, gossypol can react with other compounds to form bound
gossypol. Gossypol analyses using chemical methods do not always agree with its
bioavailability to animal possibly because of various bound forms of gossypol with
different stabilities in vivo and the (+/-)-gossypol enantiomers with different biological
activities. The objectives of this research were to produce anti-gossypol antibodies and
use antibodies to study gossypol chemistry and to analyze gossypol in cottonseed
samples and cottonseed by-products.
Polyclonal antibodies were produced from rabbits after they were immunized
with gossypol-LPH (.Limulus polyphemus hemolymph). Monoclonal antibodies were
developed by fusion of NS-1 myelomas and immune mouse splenocytes, and the
hybridomas secreting anti-gossypol antibodies were selected and cloned.
Immunoassay parameters were evaluated for their effects on antibody capture
noncompetitive ELISA (ACN-ELISA). It was found that microtiter plates, time and
temperature of incubation for antibody-antigen interactions, and reagent concentrations
for dilution of both primary and secondary antibodies could greatly influence the signal
to noise ratios in ACN-ELISA.
(+)- Gossypol and (-)-gossypol isomers were isolated by crystallization from
acetone and derivatized with L-(+)-lysine and L-2-amino-l-propanol. (+/-)-Racemic
gossypol was derivatized with L-(+)-lysine, L-(+)-arginine, ethanolamine, L-2-amino-l-
propanol, 3-amino-1-propanol, L-glutamine, L-(-)-tryptophan, L-tyrosine and para-
amino-benzoic acid.
xi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Different forms of gossypol were used in an antibody capture competitive-
ELISA to evaluate antibody sensitivity and specificity. Approximately 0.2 pg/ml to
over 125 pg/ml of gossypol equivalents caused 50% inhibition of binding (I 50) of rabbit
PAb to the solid-phase, and I 50 values ranged from 7.4 to 55 pg/ml for MAb cell lines,
when (+/-)-racemic gossypol derivatives were used competitively. I 50 values from 7.5
to 13.5 pg/ml were obtained for (+)- and (-)-isomeric gossypol derivatives as
competitors for MAb. Underivatized gossypol as competitor gave much higher I 50 value
for PAb and showed no competition for MAb.
Polyclonal antibodies were evaluated for gossypol analyses of cottonseed
products in an antibody capture competitive ELISA. The results showed good
correlation (r2=0.96) for Free Gossypol analyses and poor correlation (r2=0.43) for Total
Gossypol analyses between ELISA and AOCS official methods under the conditions of
the methods.
xii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1
INTRODUCTION AND REVIEW OF LITERATURE
1.1: Overview of cotton and cottonseed products
Cotton has long been known as nature’s unique food and fiber plant. It is
produced worldwide in tropical and subtropical regions. The People’s Republic of
China is the largest producer of cottonseed, followed by the United States and India
(Table 1).
Table 1. Cottonseed production (million metric tons) and main producers (USDA, 1998a).
Cottonseed (million metric tons) Countries Average Forecast 91/92-96/97 1997/98
China 8.05 7.72
United States 6.16 6.60
♦FSU-12 3.69 3.15
India 4.57 5.33
Pakistan 3.29 3.05
Brazil 0.86 0.65
Others 6.67 8.57
*FSU-12 represents former Soviet Union-12
World demand for cotton is growing at the rate of about two million bales a year
and if the U.S. maintains its share of world production, the U.S. will be producing about
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 million bales by the year 2000. This trend in cotton production also applies to
cottonseed production. Cottonseed ranks second to that of soybean in terms of world’s
oilseed production, but cottonseed oil only ranks sixth in world production of edible oils
because some cottonseeds are used for animal feed, such as feed for cattle and dairy
cows (USDA, 1998b).
Cottonseed, like other oilseeds, is processed by either expression or extraction
methods. There are four different milling methods that have been used for cottonseed:
(1) hydraulic pressing uses the high-pressure expression to remove oil, (2) screw
pressing is another mechanical expression method, which produces high temperature
that renders protein less soluble, (3) prepressing-solvent extraction is a combine action
of modified screw pressing and solvent extraction process that can supply a better
quality of meal proteins than screw processing, and (4) direct solvent extraction
removes the oil by using hexane or other solvents and provides a high quality protein
feed.
The composition of cottonseed, which includes oil, protein, carbohydrates,
phosphorous compounds and minerals, varies considerably depending on species,
variety, and environment. Oil is embedded in the tissue of cottonseed as droplets
(Markman, 1968).
Cottonseed averages about 45% hull and linters, and 55% kernel. The
cottonseed kernel is a pointed ovoid body approximately 8-12 mm in length, in which
there are innumerable dark spots. These dark spots are pigment glands and are unique
to cottonseed. The major component of the pigment glands is free gossypol. The
processing method affects the quantity of free gossypol and the protein value of
2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cottonseed meals (CSM). During screw pressing, most of the free gossypol binds to
amino acids of proteins and other compounds, which lowers the nutritional value of the
protein. The newer methods such as prepress solvent extraction and direct solvent
extraction produce CSM with high quality protein, but they considerably increase the
content of free gossypol.
The presence of gossypol limits the application of cottonseed protein for food
and feed. In the United States, any cottonseed protein products intended for human use
must contain no more than 450 ppm free gossypol as set by FDA (1974). The Protein
Advisory Group of the United Nations Food and Agriculture and World Health
organizations (FAO/WHO) has set limits of 600 ppm of free gossypol and 12,000 ppm
total gossypol for human consumption. Also, some caution was suggested when using
both cottonseed meal and whole cottonseed in dairy rations due to the presence of the
yellow polyphenoiic pigment gossypol (Poore and Rogers, 1998).
1.2: Gossypol
1.2.1: Occurrence of gossypol
Gossypol was first discovered and isolated as a crude pigment by Longmore in
1886 from cottonseed oil foots, a mixture of precipitated soaps and gums produced in
the refining of crude cottonseed oil with sodium hydroxide.
The chemical formula for gossypol C 30H30O8 was first established by Clark in
1927. The whole structure of gossypol was verified in 1958 by Edwards when gossypol
was synthesized and shown to be 1,1', 6,6', 7,7'-hexahydroxy-3,3'-dimethyl-5,5'-
diisopropyl-2,2'-binaphthyl-8,8'-dialdehyde (Figure 1). A comprehensive review of the
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 00
X Figure Chemical 1. structure of gossypol O
ua CO in.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. discovery, determination of structure, and chemistry of gossypol was published by
Adams et al. (1960) and Markman (1968).
Gossypol, as it exists in cottonseed, is contained in pigment glands and
constitutes 20-40% of their weight and results in 0.4 to 1.7% of whole kernel. Gossypol
glands are found not only in the seeds of cotton but also in some other parts, such as the
bark of cotton plant roots, leaves, seed hulls, and flowers. However, the gossypol
content varies widely, depending on the species and varieties of cotton plant, on
climatic and soil conditions of the region, water supply, agrotechnical treatment, and in
particular, on the amount and composition of fertilizers used (Markman, 1968;
Stansbury et al., 1954). The changes of gossypol content during different stages of
maturity also have been reported (Caskey and Gallup, 1931; Gallup, 1927, 1928). The
gossypol content of cotton plant is of importance chiefly in related to some of its unique
characteristics and its effect on the use of cottonseed meal as an animal feed, the oil in
food products, and the cottonseed flour for human consumption.
1.2.2: Physiochemical properties of gossypol
Gossypol is a chiral molecule because of steric hindrance to rotation about the
intemaphthyl bond. Cass et al. (1991) measured gossypol enantiomer ratio by HPLC
analysis after conversion to the Schiff base diastereoisomers and found that an
enantiomeric excess of (+)-gossypol was in each variety of Gossypium arboreum,
G.herbaceum and G.hirsutum, and (-)-gossypol was in excess in each variety of
G.barbadense investigated. The results agreed with that from Zhou and Lin (1988).
Gossypol contains polar groups (6 hydroxyl and 2 aldehyde groups) making it
soluble in most organic solvents, such as methanol, ethanol, isopropanol, butanol,
5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ethylene glycol, dioxane, diethyl ether, acetone, ethyl acetate, chloroform, carbon
tetrachloride, ethylene dichloride, phenol, pyridine, melted naphthalene, and heated
vegetable oil. It is less soluble in glycerine, cyclohexane, benzene, gasoline, and
petroleum ether. The presence of 2 heavy dialkylnaphthalene groups makes it insoluble
in water (Markman, 1968).
In 1960, Adams et al. proposed that gossypol existed in three tautomeric forms:
aldehyde (a), hemiacetal (b) and ketol (c) (Figure 2) in different solvents. The use of
nuclear magnetic resonance (NMR), mass spectral analysis and UV spectrometry has
illustrated its structure changes in various solutions. In basic solvent systems, gossypol
existed mainly as ketol, and in ordinary inert solvents and acidic conditions, gossypol
existed mainly in the aldehyde forms (Stipanovic, et al., 1973), while in polar solvents
such as dimethyl sulfoxide (DMSO) with alkali condition, the hemiacetal form occurred
in dynamic equilibrium with the aldehyde form (Abdullaev et al., 1990).
Gossypol is markedly reactive due to the reactivity of carbonyl and phenols, so
it has been the compound of the greatest concern in cottonseed. When removing the oil
from the cottonseed, most gossypol reacts with other compounds in cottonseed. This
results in undesirable color and a low value protein in cottonseed meal. Some gossypol
will remain in the oil. This will be removed during refining but will increase the oil
waste (Jones, 1979; Pons et al., 1951).
In order to better describe gossypol in cottonseed products, the terms “Free
Gossypol”, “Bound Gossypol” and “Total Gossypol” are used, respectively. Free
Gossypol (FG) is defined as gossypol and gossypol derivatives in cottonseed products
that can be extracted by 70% aqueous acetone (AOCS, Ba 7-58). Bound Gossypol
6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure Figure 2. Tautoniers of gossypol
7
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (BG), formed during cottonseed processing by reaction of gossypol with other
compounds, is not soluble in aqueous acetone. Total Gossypol (TG) is defined as
gossypol and gossypol derivatives, both FG and BG, that can react with 3-amino-1-
propanol in dimethylformamide solution (AOCS, Ba 8-78). This AOCS method
(AOCS, Ba 8-78) only measures gossypol, gossypol analogs and gossypol derivatives
that have an available aldehyde moiety for the derivatization reaction. It should be
noted that there might be a fraction of gossypol that is unavailable for this reaction and
therefore is not included in the TG value. FG and TG are determined empirically, and
BG is determined mathematically (BG=TG-FG)
Some evidence indicates that the majority of gossypol is in the form of Schiff
bases from the condensation between aldehydic groups of gossypol and e-amino groups
of proteins during cottonseed processing (Cater, 1968; Lyman et al., 1959). However,
this chemical complex alone can not fully account for gossypol behavior. Gossypol
may be chelated by iron in cottonseed products to form insoluble metal complexes
(Muzaffaruddin and Saxena, 1966), may form gossypol polymers (Anderson et al.,
1984), or may be oxidized (Scheiffele and Shirley, 1964). The phenolic groups may
react to form esters and ethers with other carboxylic compounds and phenols in
cottonseed plants. Two types of reactive functional groups in gossypol: aldehydes and
phenols, may give rise to several forms of bound gossypol.
In past years, much work has been done on the Schiff base reaction between
gossypol and amino groups. During cottonseed processing, the moist heat treatment of
cottonseed kernels catalyzes the chemical reaction between gossypol and the free amino
groups of cottonseed protein. Soluble gossypol (FG) in aqueous acetone was thus
8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. converted to insoluble Bound Gossypol (BG). Baliga (1956) found that lysine accounted
for most of the free amino groups of cottonseed protein and that reaction of gossypol with
cottonseed protein occurred mainly with e-amino groups of lysine, thus the lysine
availability decreased to about one-half of the original value (82.9% to 48.7%) when the
purified protein was allowed to react with gossypol. Involvement of arginine in the
reaction has also been reported (Martinez et al., 1961). In 1979, Damaty and Hudson
studied the chemical nature of interaction using selective proteolysis, gel filtration and
amino acid analysis and concluded that lysine and the more hydrophobic amino acid in
cottonseed protein were easily to take part in the formation of insoluble material. In 1988,
Reddy and Rao studied the interaction between gossypol and gossypin, congossypin and
glycinin using a difference spectral method and found that the interaction was completely
reversible, and suggested that hydrophobic and ionic interactions were involved in the
reaction of gossypol with proteins. Later, Strem-Hansen et al. (1989) studied the
interaction of gossypol with amino acids and peptides using circular dichroism (CD) and
nuclear magnetic resonance (NMR) and gave evidence that hydrophobic interaction may
be responsible for a significant proportion of the interaction between gossypol and
proteins. This opinion was also supported by the finding that gossypol bound
competitively at the bilirubin binding site on albumin (Royer and Vander Jagt, 1983). This
site is known to be linked with many hydrophobic residues, with only one or two
positively charged amino acids being present. The total binding energy of the complex
would be the sum of the hydrophobic interaction and Schiff base formation.
Another investigation (Lyman et al., 1959) of gossypol-protein complexes
revealed that when gossypol combined with crystalline BSA or cotton protein, the point of
9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. attachment is the e-amino group of lysine. The molar ratios of free e-amino groups to
gossypol varied according to the experimental conditions, but averaged about 1.5 moles of
lysine being bound to 1 mole of gossypol. At low concentrations of gossypol, the molar
ratio of lysine to gossypol was 2:1, indicating that both of the reactive aldehyde groups of
gossypol were linked to lysine. Sedimentation velocity studies of these gossypol-protein
complexes suggested the presence of from 1 to 4 different compounds, thus indicating that
gossypol probably formed a cross-link between two or more protein molecules.
Later, model complexes of gossypol with amino acids (lysine, asparagine,
glutamine, and glycine), peptides (hippuryl-L-lysine, L-alanyl-L-lysine, glycyl-L-lysine,
L-histidyl-L-lysine) and purified proteins (glandless cottonseed protein, cottonseed
globulin, insulin) were synthesized and partially characterized (Cater, 1968), and it was
concluded that the rate of reaction of gossypol with amino acids increased with an increase
of pH (5.7 to 7.5), and was shown to be related to the distance of the amino group from the
carboxyl group within molecule ( e.g. the e-amino group is more reactive than an a-amino
group of an amino acid). Whaley et al. (1984a and 1984b) studied the reaction of (+)-
gossypol with BSA, human serum albumin, lactate dehydrogenase, malate dehydrogenase,
alkaline phosphatase, lysozyme, protamine and poly-L-lysine and measured the products
using circular dichroism (CD) and found that (+)-gossypol bound to albumin with the
same affinity as (+/-)-gossypol. The reactions between gossypol and BSA, glandless
cottonseed meal and glanded cottonseed meal also were studied by Couch and Thomas
(1976).
A Schiff base is a relatively labile bond that is readily reversed by hydrolysis in
aqueous solution. It can be chemically stabilized by reduction. The formation of a
10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Schiff base is enhanced at alkaline pH values, but is still not completely stable unless
reduced to a secondary or tertiary amine linkage (Hermanson, 1995). A number of
reducing agents can be used to convert the Schiff base into a secondary amine. The
addition of sodium borohydride or sodium cyanoborohydride will result in reduction of
the Schiff base intermediate into a relatively stable secondary amine. Both borohydride
and cyanoborohydride have been used for reductive amination purposes, but
borohydride will reduce the reactive aldehyde groups to hydroxyls at the same time
when it converts Schiff bases present to secondary amines. Cyanoborohydride, by
contrast, is a milder reducing agent that is at least five times milder than borohydride in
reductive amination processes. Cyanoborohydride does not reduce aldehydes, but it is
very effective for Schiff base reduction (Lane, 1975; Hermanson, 1995). Thus, higher
yields of conjugate formation can be obtained using cyanoborohydride instead of
borohydride for the stabilization of Schiff base products.
The available lysine in BSA, glandless cottonseed meal, solvent extracted
cottonseed meal and gossypol complexes of these materials has been determined by
different chemical methods, such as the dinitrofluorobenzine (DNFB), sodium borohydride
(NaBH*), and trinitrobenzenesulfonic acid (TNBS) methods. However, it was found that
different methods could give different results (Couch and Thomas, 1976). By measuring
available lysine, Anderson et al. (1984) found that most gossypol was attached to other
groups (not lysine) in the proteins or had formed large gossypol polymers, binding to very
few lysine groups. This opinion is different from previous reports (Baliga, 1956; Cater,
1968; Lyman etal., 1959; Martinez etal., 1961).
11
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The reactions have also been studied between gossypol and other amine
compounds such as para-amino-benzoic acid, y-amino butyric acid, anisidine and (+)-l-
phenylethylamine. Interested readers can find the related articles published by Gdaniec
(1994) and Cater (1968).
1.2.3: Utilization and physiological properties of gossypol
Gossypol is one of the secondary metabolites defined as compounds that are not
ubiquitous and that do not play an indispensable role in plants that synthesize them. In
the natural environment, plant phenolic metabolites play an important role as
allelochemicals. They function as signals between plants, between plants and
symbiontic or pathogenic organisms to protect plants against microbes or insects
(Heller, 1993). Being a polyphenolic compound, gossypol is an anti-oxidant
(Mar km an, 1968).
Gossypol and many of its derivatives are reactive, so gossypol may serve for the
synthesis of various useful organic compounds with unique structures. For example,
gossypol pitch, obtained from cottonseed oil soapstock by distillation, is used in the
production of core binder agent to improve road surfaces. In addition, taking advantage
of its optically active nature, the (+)-enantiomer of gossypol has been shown to be a
useful CD (circular dichorism) probe to study interactions with human and bovine
serum albumin (Whaley et al., 1984a and 1984b).
Gossypol also has a great number of pharmacological activities. Gossypol has
been studied extensively as a potential male contraceptive agent in several mammalian
species, including rats (Hadley et al., 1981; Lin et al., 1980; Lin et al., 1985), mice
(Coulson et al., 1980), monkeys (Shandilya et al., 1982), hamsters (Matlin et al., 1985;
12
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Waller et al., 1984), rabbits (Chang et al., 1980), bulls (Arshami and Ruttle, 1988) and
human (National Coordinating Group on Male Antifertility Agents, 1978). The basis of
this gossypol-induced infertility is reduction in sperm counts, abnormal sperm structure,
and loss of sperm forward motility (Chang et al., 1980; Lin et al., 1980; Shandilya et al.,
1982) and it was found that (-)-gossypol is more active in antifertility function than (+)-
gossypol (Matlin et al., 1985).
Lin et al. (1989 and 1993) reported that gossypol inhibited the replication of
human immunodeficiency virus-type 1 (HIV-1) and found the (-)-enantiomer of
gossypol to be more inhibitory compared to the (+)-enantiomer. Gossypol is capable of
inhibiting the growth of a variety of cell lines derived from epithelial tumors (Benz et
al., 1988; Benze et al., 1991; Wu et al., 1989), breast carcinoma and esophageal cancer
(Hu et al., 1994; Gilbert et al., 1995) and transplantable tumors (Rao et al., 1985).
These disruptions include inhibition of cytoplasmic and mitochondrial enzymes
involved in energy production (Ueno et al., 1988) and uncoupling of oxidative
phosphorylation (Flack et al., 1993; Abou-Donia and Dieckert, 1974). Depletion of
cellular adenosine triphosphate (ATP) has been demonstrated in cultured tumor cells
(Keniry et al., 1989). Gossypol also inhibits key nuclear enzymes responsible for DNA
replication and repair, including DNA polymerase a (Rosenberg et al., 1986) and
topoisomerase II, and block DNA synthesis in HeLa cells (Wang and Rao, 1984).
These properties make gossypol a potential antineoplastic agent.
Gossypol also can inhibit glutamic, malic and alcohol dehydrogenases. It was
found that (-)-gossypol is 13 times more active in inhibiting the enzyme than (+)-
gossypol and (-)-gossypol is 3 times more active in inhibiting amebic culture growth
13
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Gonzalez-Garza et al., 1992; Gonzdlez-Garza et al., 1993). The studies on the
inhibition of rabbit sperm acrosomal enzymes by gossypol indicated that azocoll
proteinase, acrosin, neuraminidase, and aryksukfatase were significantly inactivated by
gossypol at 12-76 pM, and hyaluronidase, (i-glucuronidase, and acid phosphatase were
inhibited at higher concentration of gossypol at 380 pM (Yuan et al., 1995).
Gossypol has general antifimgal activities with LD 50 values from 20 to 100 ppm
of pure gossypol (Bell, 1967) and has inhibitory effect on microorganisms including
aerobic sporeformers and lactobaciili and some yeasts (Margalith, 1967). It also has
been suggested to be of value in the treatment Chagas disease (Montamat et al., 1982).
Another important aspect of gossypol and its derivatives is that they could lower plasma
cholesterol levels (Shandilya et al., 1982). In 1994, Acheduma et al. reported that
gossypol and related derivatives might in some way contribute to the lowering of
cholesterol level in rats. Later, Nwoha (1995) measured serum constituents of gossypol
treated, protein-malnourished Wistar rats and found that the administration of gossypol
decreased the levels of serum cholesterol.
1.2.4: Toxicity and detoxification of cottonseed products
Cottonseed is an important protein supplement for livestock. Cottonseed meal, a
by-product of the cottonseed oil industry, contains about 40-50% crude protein and is a
good protein resource for cattle. Cottonseed has been utilized as a feed for livestock
since the early 19th century.
A number of investigators have indicated that gossypol is toxic to monogastric
animals and also to young ruminants. The most common toxic effect of gossypol is
cardiac irregularity which causes death of the animal, because gossypol prevents the
14
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. liberation of oxygen from oxyhemoglobin (Menaul, 1923). The toxicological effects of
gossypol have been classified in three levels, which are: ( 1 ) acute doses causing
circulatory failure, (2) subacute doses causing pulmonary edema, and (3) chronic doses
causing symptoms of ill health and malnutrition (Abou-Donia, 1976). The doses for
each level are different for different species.
Since gossypol exhibits this dose response, there are some restrictions for the
safe use of cottonseed meal as livestock feed. A study using male rats (Eagle and
Davies, 1958) showed that oral LD 50 is from 1061 to 2170 mg/kg administering
gossypol as cottonseed pigment glands (27.0 to 37.8% of gossypol), and oral LD 50 is
from 2200 to 2600 mg/kg with pure gossypol (ca 100% gossypol). The cottonseed
pigment glands seemed more toxic than pure gossypol for rats in this study. El-
Nockerashy et al. (1963) had similar findings in studying the acute oral toxicity for rats
by feeding cottonseed pigment glands, gossypol, diaminogossypol and gossypurpurin
with LD50 1120, 2570, 3270 and 6680 mg/kg body weight, respectively. Calhoun et al.
(1990b) compared toxicity of gossypol acetic acid and Free Gossypol in cottonseed
meal and pima cottonseed by feeding lambs, and found that oral administration of
gossypol acetic acid is much more toxic to young lambs than feeding equivalent
amounts of Free Gossypol from cottonseed meal or cottonseed. Gossypol in different
status may have different stabilities in the whole digestive tracts of either ruminants or
nonruminants. Gossypol in unbroken glands may be protected by the gland envelope,
and the other ingredients in the diet may also affect the gossypol stability. For rainbow
trout (Herman, 1970), 95 ppm of Free Gossypol in the diet caused histological changes
in the liver and kidney; 100 ppm could cause pathological changes; 290 ppm could
15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. suppress growth; at 531 ppm level, fish lost weight for a period of time and suffered
severe reduction of hematocrit, hemoglobin and plasma proteins. A study using channel
catfish (eight weeks old) showed that diets containing approximately 0.14% Free
Gossypol from cottonseed meal or gossypol acetate added in meal could depress
growth. A level of 0.09% or less Free Gossypol in the diet seemed to be safe if all
essential amino acids were in balance (Dorsa and Robinnette, 1982). Swine showed
toxicosis when fed 200 to 400 ppm Free Gossypol in the diet and it was suggested that
Free Gossypol should be less than 100 ppm for growing and fattening swine (Haschek
etal., 1989).
Cattle were found to be more tolerant to gossypol than young calves due to the
action of the rumen (Morgan, 1989). Lindsey et al. (1980) studied the physiological
responses of lactating cows to gossypol from cottonseed meal and suggested that
detoxification occurred in mature ruminants consuming cottonseed meal containing
high Free Gossypol. It was demonstrated that rumen microorganisms were responsible
for the detoxification of gossypol and it was proposed that gossypol formed a gossypol-
protein complex (gossypol-microbial protein) with soluble protein in the rumen liquor
which was not absorbed in the digestive tract. This complex was very stable during
enzymatic hydrolysis (Reiser and Fu, 1962). The microbial population in the rumen is
regulated by the ecological balance of conditions that tend to prevail there (Van Soest,
1982). The higher microbial yield characteristic of the rumen may be important in
supplying enough protein to capture the gossypol.
However, if gossypol content is too high in the diet, it will suppress the rumen’s
ability to detoxify. It was found that 348 to 414 mg of Free Gossypol per day would
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cause congestive heart failure in adult goats (East et al., 1994) and 8 mg free and 222mg
of Total Gossypol (in extracted cottonseed meal)/kg body weight per day could cause
physical and hematological changes at 2 weeks for cows (Lindsey et al., 1980).
Because of the toxicity of gossypol to animals, a glandless variety of cotton was
developed in the early 1960s. It was believed that the value of cottonseed oil and meal
would be improved if gossypol were not present. However, this glandless strain is
easily susceptible to insect attack, and attracts field mice and other rodents (Stipanovic
et al., 1986; Lusas and Jividen, 1987). Dilday (1986) developed a cotton plant through
an interspecific cross of tetraploid (2N=52) Gossypium hirsutum x diploid L. (2N=26)
G. sturtianum Willis. This plant showed gossypol glands in vegetative foliar and
fruiting tissues but not in the seed. However, this approach needs study to prove the
stability and yeild for this plant.
Another approach for gossypol removal is by a flotation process based on the
specific gravity difference or by centrifugal force, such as liquid cyclone process which
is a physical separation of intact pigment glands (Gardner et al., 1976).
Chemical methods are commonly used methods for gossypol detoxification.
Bressani et al. (1964) reported that an alkaline pH of cooking, associated with calcium
ions, was important in reducing Free Gossypol and Total Gossypol in cottonseed flour
used for human foods. The addition of calcium increased the effectiveness of the
gossypol-iron complex formation, resulting in full protection from gossypol toxicity.
Iron sulfate is an inexpensive source of Fe that could also reduce gossypol intoxication
in nonruminant animals (Ullrey, 1966).
17
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Kemmerer et al. (1966) reported that addition of iron sulfate to feed could
prevent yolk discoloration in cold-stored eggs caused by gossypol. Muzaffaruddin and
Saxena (1966) showed that a 1:1 molar ratio of ferric iron to gossypol formed an iron
gossypol complex, in which the two perihydroxyl groups of gossypol were the most
plausible sites where the ferric irons chelated. Barraza et al. (1991) investigated the
efficacy of iron sulfate and feed pelleting to detoxify Free Gossypol in cottonseed diets
for dairy calves. Ferrous sulfate added to diets for swine contained 244 and 400 ppm
Free Gossypol (FG) at a molar ratio of 0.5:1 for iron to gossypol, gave partial
detoxification and a 1:1 ratio gave complete detoxification. The addition of
phospholipids in cottonseed meal or cottonseed meat coupled with cooking could
eliminate some Free Gossypol (FG) and improve the protein quality (Yannai and
Bensal, 1983).
Recently, Calhoun (1999) and Wan (1999) studied the effects of different
processing methods on the gossypol availability through feeding lambs and cattle.
Gossypol toxicity was influenced by the methods of cottonseed processing, dietary
concentrations of iron, calcium, and the presence of other toxic terpenoids and
chemicals found in cottonseed, as well as the amount and duration of gossypol
consumption.
It has been believed for a long time that BG is physiologically inactive and FG
is physiologically active. The FG content of cottonseed products however is not a good
predictor of the cottonseed product toxicity (Calhoun et al., 1990a; Calhoun, 1996; Eagle
and Davies, 1958; Eagle et al., 1956), and the measurements of BG, FG and TG (Total
Gossypol) are also insufficient. We would like to propose the BG fraction is not always
18
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. entirely inactive depending on the mechanism(s) of gossypol binding that occurs during
cottonseed processing, the type of animal consuming the gossypol and physical form of
the feed.
1.2.5: Gossypol analysis
Several methods for the quantitative determination of gossypol in cottonseed, oil,
press-cake, and meal have been reviewed by Markman (1968). These include gravimetric
methods, volumetric methods, colorimetric, spectrophotometric, polarographic and
luminescent methods.
Gossypol can be derivatized to form a trimethylsilyl (TMS) derivative and
determined by gas-liquid chromatography (Raju and Cater, 1967). Near-infrared
reflectance, which has been used in many applications for composition analysis (Norries
et al., 1976), also offers a possibility for the measuring gossypol content (Birth and
Ramey, 1982). The structural analysis of gossypol, gossypolone and correlated
derivatives were studied using electron impact-mass spectrometry (EI-MS) and infrared
spectrometry. Mass spectra and IR spectra obtained from these derivatives may aid in
the identification of compounds that are found in association with gossypol (Phillip and
Hedin, 1990).
The most common methods for gossypol analysis include high performance
liquid chromatography (HPLC) (Botsoglou, 1991; Chamkasem, 1988; Hron et al., 1990;
Stipanovic et al., 1988) and AOCS official methods (AOCS, Ba 7-58 and Ba 8-78).
Both methods measure either “Total Gossypol” or “Free Gossypol” defined by AOCS
official methods (Ba 7-58 and Ba 8-78). Total Gossypol (TG) is gossypol and gossypol
derivatives, both FG (Free Gossypol) and BG (Bound Gossypol), in cottonseed products
19
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that can react with 3-amino-1-propanol in dimethylformamide solution to form a
diaminopropanol complex. Free Gossypol (FG) is gossypol or gossypol analogs which
can be extracted by 70% aqueous acetone. High performance liquid chromatography
(HPLC) has been used to determine gossypol and other individual terpenoid aldehydes
in seed, leaves and flower buds, and in processed oils and meals (Chamkasem, 1988;
Hron et al., 1990; Nomeir and Abou-Donia, 1982; Stipanovic et al., 1988). The
gossypol determined using HPLC is not correlated with the results obtained from AOCS
official methods. The AOCS official method measures all the gossypol and gossypol
derivatives having available aldehydic groups. The AOCS official method may lead to
false readings due to the presence of non-gossypol aldehydic-containing compounds
that react with coloring agent (Stipanovic et al., 1984).
Most toxicity studies for animals with gossypol have been based on the AOCS
official methods and HPLC methods. Some questions exist about the validity of
gossypol analytical procedures because of inconsistencies between determined gossypol
content and the biological activities of the samples (Calhoun et al., 1990a; Calhoun,
1996; Eagle and Davies, 1958; Eagle, et al., 1956). One explanation for this
phenomenon could be the association of various compounds with Free Gossypol
yeilding different physiological activities (Calhoun et al., 1990a; Calhoun, 1996; Eagle
and Davies, 1958; Eagle, et al., 1956), and the existence of (+/-)- two gossypol
enantiomers, having different biological activities. Another reason may be that
different forms of bound gossypol have different stabilities in vitro and in vivo
(Calhoun, 1996), possibly because different cottonseed processing conditions favor
20
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. various forms of bound gossypol. The animals selected for bioactivity experiments also
would affect the results because of differing digestive environments.
Results related to available gossypol must be evaluated and interpreted
carefully, considering the dose and route of administration, the possibility of other toxic
materials, and the degree of inactivation of administered gossypol before and/or after
administration by reaction with components of the diet (Yu, 1987).
Because of the complicated reactivity of gossypol in cottonseed products and its
apparently variable physiological properties, specificity and accuracy of gossypol
analysis becomes more important. This is especially important when one considers
potential new applications. At the same time, the determination of FG, BG and TG is
insufficient, and it is also necessary to determine the forms in which gossypol exists and
their relationship with bioactivity.
For the reasons explained above, gossypol screening requires an improved
analytical method that could facilitate fast and accurate determination and improve the
management, study and utilization of cottonseed products for feed or food. Because of
the selectivity of antibody binding, an immunochemical method would possibly
simplify and improve current gossypol analyses. The acceptability of immunoassays is
increasing for pesticide and other environmental analyses (Abouzied et al., 1993; Chen
et al., 1995). The antibody specific binding make it possible to analyze crude extracts,
possibly both soluble and insoluble analytes such as matrix-bound gossypol. A low
detection limit is another advantage because this allows one to analyze a small sample
that has been sufficiently diluted to remove any interfering compounds. Monoclonal
antibodies (MAb) may be able to distinguish different forms of bound of gossypol and
21
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (+/-)-isomers of gossypol, and therefore an assay based on these antibodies may provide
greater accuracy of analysis.
1.3: Theory of enzyme immunoassay
1.3.1: Enzyme immunoassay
Immunoassays are analytical methods that are based on the specificity of
antibody-antigen (Ab-Ag) interaction. There are two classes of immunoassays
including isotopic immunoassay such as radiaoimmunoassay (RIA) and nonisotopic
immunoassay including fluoroimmunoassay (FIA) and enzyme immunoassay (El)
(Dixon, 1994). Isotopic immunoassay is based on competition for antibody between a
radioactive indicator antigen and unlabeled antigen in the test sample, and nonisotopic
immunoassays are different from isotopic immunoassays, mainly due to the type of
label used, and the means of endpoint detection. Enzyme immunoassays employ
enzyme markers as means of amplifying and visualizing the primary Ab-Ag binding
reaction. This method is widely used in quantitative and qualitative determination
(Nakamura et al., 1986). Enzyme tags are easy to handle, relatively inexpensive and
stable. Also, they can convert a colorless substrate to a colored product giving sensitive
and easy to interpret endpoints. This method, when one of the binding elements is
attached to a solid support, is termed an enzyme-linked immunosorbent assay (ELISA).
The use of a solid support enables easy separation of the unbound fraction from the
bound fraction.
An Ab capture indirect competitive ELISA will be used in this study. The basic
steps involved in this assay are shown in Figure 3. First, a standard amount of antigen
is immobilized on the surface of microtiter plate. Then, sample antigen to be analyzed
22
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A____ A
Sample Ab AA B
Enzyme labeled 2nd Ab
Enzyme substrate
Figure 3. Antibody capture competitive ELISA (enzyme linked immunosorbent assay) A. Ag is immobilized on a plastic surface. B. Competition occurs for Ab binding sites between the immobilized Ag and added sample Ag. C. After removing unbound materials, the Abs bound to immobilized Ag are detected by adding peroxidase labeled 2nd Ab. D. After removing excess reagents, peroxidase substrate is added and color is developed.
23
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or standard solutions along with a known amount of the antibodies are added. Free
antigen competes with immobilized antigen for antibodies. When the free antigen
concentration is higher, less Ab will bind to immobilized antigen. After washing away
the unbound materials, all remaining antibodies are detected by adding enzyme labeled
2nd Ab (e.g. goat anti-rabbit IgG and goat anti-mouse IgG+IgM, conjugated to enzyme).
After removing the excess reagents, enzyme substrate is added, and color is measured.
The amount of color produced is inversely proportional to amount of antigen from the
sample or standard solution. The color measured is greatest, when free antigen is 0. In
this case, the assay is called Ab capture indirect non-competitive ELISA.
1.3.2: Antibody structure
Antibodies are globular proteins commonly referred to as immunoglobins (Ig).
Structurally, antibodies are composed of one or more copies of a characteristic unit that
can be visualized as a Y shape. The unit consists of four polypeptide chains: two
identical heavy chains and two identical light chains (Figure 4). Each arm containing a
heavy chain and light chain, contains a site that is responsible for antigen binding. The
variable regions of the amino acid sequence in this portion provide the basis for the Ab-
Ag binding and are responsible for Ab specificity (Rittenburg, 1990).
Antibodies can be divided into five classes, IgA, IgG, IgE, IgM, and IgD based
on their structures. Among the immunoglobins above, IgM is the first antibody
produced when an animal is exposed to an immunogen and it exists as a pentamer in
serum. IgG is a monomer that is normally produced later in the immune response, it
makes up about 89% of the total Ig in the serum, and it is the most common type of
antibody employed in immunoassay (Rittenburg, 1990).
24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure Figure 4. Antibody basic unit structure
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.3.3: Antibody-antigen (Ab-Ag) interactions
The interaction between Ab and Ag is noncovalent and reversible. The binding
includes hydrogen binding, electrostatic, Van Der Walls and hydrophobic interactions.
The binding of antibody to antigen follows basic thermodynamic principles of any
reversible biomolecular interaction. The equilibrium equation can be defined as:
_ [Ab- Ag]
eq ‘ [Ab]\Ag]
Where Keq is the equilibrium constant; [Ab], [Ag], and [Ab-Ag] are the molar
concentrations of the free Ab, free Ag and Ab-Ag complex in the equilibrium status,
respectively. For the reversible equilibrium reaction, temperature, pH, and buffers
affect the affinity constant, Keq. The time taken to reach equilibrium depends on the
diffusion rate.
1.3.4: Polyclonal antibody (PAb) and monoclonal antibody (MAb) production
When higher vertebrates are exposed to foreign materials, antibodies are elicited
as part of their immune response. The immune response involves many interactions of
a large numbers of specialized cells: macrophages, T lymphocytes and B-lymphocytes
(Harlow and Lane, 1988b). When an animal is exposed to an immunogen with an
appropriate immunization schedule, B cells will be activated and will proliferate and
differentiate into plasma and memory cells. Plasma cells produce large amounts of
antibody. Memory cells are long-lived cells that remain in circulation and are primed
upon subsequent exposures to the antigen. The differentiation of B cells leads to the
production of higher affinity Ab, when the animal is exposed to the antigen again.
26
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Considerations need to be made before immunization, such as choice of animal,
form of immunogen, type of adjuvant, dose and boosts. Gossypol (MW 518) is not
large enough to elicit Ab, so it must first be conjugated to a carrier protein to become an
immunogen. The ability of the immunogen to elicit the desired immune response
depends on the amount of hapten as well as its orientation. The presence of spacer alkyl
chains, such as e-amino group of lysine induces a greater immune response. Too long
or too short spacer arms would limit the exposure of the analyte for antibody production
and decrease the assay sensitivity (Chen et al., 1995). The purified immunogen
combined with an immune stimulant (adjuvant) can enhance the immune response. The
application of adjuvants could slow down the release of immunogen, give rise to a high
antibody response and induce an increased circulation of lymphocytes to allow greater
contact between antigen and antigen-reactive cells to increase the antibody production
(Harlow and Lane, 1988a). Boosts can improve the maturation and differentiation of B
cells to produce high affinity antibodies.
If blood from an immunized animal is the source of Ab that contains a
heterogeneous population of Ab, this is referred to as polyclonal antibodies (PAb).
Each antibody population differs in both its affinity and its specificity from others,
because each population represents the secreted products from a single stimulated
lymphocyte and its clonal progeny (Hum and Chantler, 1980). Usually rabbits are the
first choice for PAb production, since they are cheap, easy to care for, robust, and easy
to bleed. Figure 5 shows the steps involved to produce PAb by immunizing a rabbit.
Compared to PAb, MAb are more specific, because they are derived from a
cloned lymphocyte cell. MAb can be produced after immunizing mice with
27
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunogen
I
H Immunization
I
Serum collection (jl I II )
Figure 5. Diagrammatic representation of the production of PAb (polyclonal antibodies) from rabbits
28
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. immunogen. The immunized mouse spleen has a high density of B cells. Each B-cell
clone contains the genes to produce one type of antibody. The fused cells between
myeloma cells and splenocytes, called hybridoma cells, have the characteristics of both.
After fusion, the hypoxanthine-aminopterin-thymidine (HAT) system is usually used to
select the hybridomas (Kohler and Milstein, 1975). In this system, aminopterin (A) is
used to block the main biosynthetic pathway for nucleic acid production. The mutant
myelomas, lacking the enzyme hypoxanthine guanine phosphoribosyle transferase
(HGPRT), will multiply in the absence of aminopterin, but die out in HAT medium.
Normal cells can continue to synthesize nucleic acids using the salvage pathway when
hypoxanthine and thymidine are provided. Spleen cells, being primary cells, however,
will not grow very long in culture without special stimuli and conditions. Fused
hybridomas can grow in HAT medium. Selected hybridomas are screened for the
production of Ab with desired characteristics and positive cells are cloned. Each
positive cell line may have different specificity, so MAb with different specificities can
be obtained. The Figure 6 shows the basic steps for MAb production and Figure 7
shows the details of HAT selection system.
There are two different ways to produce large amounts of MAb from a secreting
hybridoma line: by growing the cell line in culture or by growing it in animal as a tumor
(Zola, 1987b). For the first method, the antibody is produced by maintaining the
hybridomas in culture and splitting the culture every 2 to 4 days. Alternatively, the
hybridoma is injected into intraperitoneal of an appropriate animal and grows as a
transplantable myeloma, secreting large amounts of immunoglobulin in peritoneal
cavity as ascitic fluid. The ascitic fluid is harvested as the source of MAb. In general,
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Immunization
£ a Splenocytes in spleen
•• Myeloma
Cell fusion and HAT selection i
fvhybridoma i ran screening i
t cloning
Ab, Ab, Ab„
Figure 6 . Diagrammatic representation of the production of MAb (monoclonal antibodies) from mice
30
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Salvage Salvage De novo nonviable (in tissue culture) HAT selection Salvage hybridoma Cell fusion De novo myeloma B cell nonviable viable Figure 7. The HAT (hypoxanthine-aminopterin-thymidine) selection system (Adapted from Harlow and Lane, 1988)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tissue culture supernatant is not contaminated by other antibodies, but can only be
produced at relatively low concentrations. Supernatants can be used for most serology.
Ascites produced by animals may be contaminated with other immunoglobulins made
by the host, but can be produced in higher quantities. Ascitic fluid is usually used for
immunochemical characterization or isolation of antigens. This is common method of
commercial MAb production.
One of the principal advantages of a hybridoma culture is that it provides a
potentially permanent source of Ab. Once a cell line of interest is obtained, the cells
can be preserved indefinitely using cryopreservation. The temperature of liquid
nitrogen is -196°C. In order to reduce damage caused by the formation of ice crystals,
cells are slowly cooled down and a cryoprotective medium (for example,
dimethylsulfoxide in serum) is used.
MAb used in immunoassay have some advantages over PAb: their specificity of
binding, their homogeneity, and their ability to be produced in unlimited quantities.
One unique advantage of hybridoma production is that impure antigens can be used to
produce specific antibodies. Because hybridomas are single-cell cloned prior to use,
monospecific antibodies can be produced after immunizations with complex mixtures of
antigens.
1.4: Research objectives
The reactivity of gossypol has made study and understanding of its nature
difficult. Besides natural (+)- and (-)-isomers, there are various forms of bound
gossypol. Different forms of gossypol with different stabilities and biological activities
lead to the inconsistant results between chemical analyses and animal availability and
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bioactivity studies. We are proposing to develop and apply immunochemical methods
for gossypol anlalysis. Because of the selectivity and specificity of Ab-Ag interaction,
immunochemical methods may provide a potential tool to study gossypol forms and
their availabilities. The specific objectives were to:
1. Produce protein-gossypol conjugates to immunize rabbits and mice for the
production of polyclonal and monoclonal anti-gossypol antibodies.
2. Use characterized antibodies to study the chemistry of gossypol interactions with
amino and amine compounds in model systems, and improve our knowledge of the
forms and bioactivity of bound gossypol.
3. Adapt and optimize the above antibodies in an immunoassay to rapidly and
accurately measure the quantity and/or quality of gossypol in conttonseed products.
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2
MATERIALS AND METHODS
2.1: Materials
Bovine serum albumin (BSA), Limulus polyphemus hemolymph (LPH),
hydrogen peroxide, ammonium carbonate, ammonium chloride, trypan blue, sodium
pyruvate (100 mM), sterile cell culture penicillin-streptomycin (with 10,000 units
penicillin and 1 0 mg streptomycin per ml), sterile fetal calf serum, oxalic acid, 2 ,2 ’-
azino-bis (3-ethylbenthiazoline-6-sulfonic acid) (ABTS), Polyoxyethylene-20-
sorbitanmonolaurate (Tween 20), N,N-dimethylformamide, ammonium sulfate,
potassium phosphate (monobasic), ethanolamine, L-2-amino-l-propanol, 3-amino-1-
propanol, gossypol, L-glutamine, L-(-)-tryptophan, L-tyrosine, para-amino-benzoic
acid, sterile dimethylsulfoxide (DMSO), cellulose dialysis tubing sacks (MWCO
12,000), agarose (type VII: low gelling temperature), goat anti-mouse IgG+IgM (H+L)
peroxidase conjugated, Rosewell Park Memorial Institute 1640 (RPMI 1640) and
AvidChrom-Protein A Antibody Purification Kit (with HEPES buffer, generated buffer,
serum-binding buffer and elution buffer) were purchased from Sigma Chemical Co. (St.
Louis, MO). Citric acid (monohydrate granular) was from Mallinckrodt Inc. (Paris,
KY). L-(+)-lysine and L-(+)-arginine were purchased from Acros Organics (Geel,
Beljium, NJ). Sodium cyanoborohydride was from Aldrich Chemical Company, Inc.
(Milwaukee, WI). Goat anti-rabbit IgG (H+L) peroxidase conjugated, Freund’s
incomplete adjuvant and Freund’s complete adjuvant were purchased from Calbiochem-
Novabiochem Corp. (La Jolla, CA). The Ribi adjuvant for mouse immunization was
34
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. purchased from Ribi Immunochem Research, Inc. (Hamilton, MT). Polyethylene glycol
1500 (PEG-1500) was purchased from Boehringer Mannheim GmbH, Germany. HT
and HAT were bought from Gibco BRL (Grand Island, NY) as lyophilized
reconstitutable as lOOx sterile solutions (10 mM sodium hypoxanthine and 1.6 mM
thymidine and with 40 pM aminopterin in HAT only). Acepromazine maleate, atropine
sulfate SA, xylazine and ketamine were provided by Animal Care Facility at the
Louisiana State University. The myeloma cell line (NS-1) was a non-secreting clone of
P3x63Ag8 (American Type Culture Collection, Rockvillie, MD).
Single frosted autoclaved microscope slides (25x75xlmm) were purchased from
Sigma Chemical Co. (St. Louis, MO). Disposable microcapillary pipets (25 (al) were
purchased from Kimble (Toledo, Ohio). Flexible polyvinylchloride (PVC) microtiter
plates were from Dynatech Laboratories Inc. (Alexandria, VA). Dynex Immulon® 1 B,
Immulon® 2 HB and Immulon® 4 HBX plates were obtained from Dynex Technologies,
Inc. (Chantilly, VA). Whatman No.l and No.2 filter papers were bought from Whatman
International Ltd. (Maidston, England). Needles (25G5/8, 18G11/2, 20G11/2) were
from Becton Dickinson Labware (Franklin Lakes, NJ). Cottonseed product samples
were gifts from M. Calhoun and sample ID number (2241, 2817, 2822, 2819, 2820,
2814 and 2813) assigned in the Nutrition/Toxicology Laboratory at the Texas A&M
University Agricultural Center. Free Gossypol and Total Gossypol were previously
measured using AOCS official methods, and (+)- and (-)-gossypoI isomers were
measured using HPLC by M. Calhoun (Texas A&M University Agricultural Center,
San Angelo, TX). Two New Zealand white (NZW) rabbits and five mice (BALB/c)
were housed and cared by the Animal Care Facility at the Louisiana State University.
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sterile plasticware used for fusion, cloning and tissue culture included 96-well
and 24-well covered tissue culture plates (Costar, Cambridge, MA), 60x15mm and
100x20mm tissue culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ), 15
ml and 50 ml disposable polypropylene centrifuge tubes, and 200pl universal pipet tips
(Coming Inc., Coming, NY), 1ml pipet tips (dot Scientific Inc., Burton, MI), sterile
solution basins (Labcor Products, Inc., Frederick, MD), 2.5ml and 12.5 ml eppendorf
tips (Brinkman Instrument Inc., Westury, NY), 0.22 pm and 0.45 pm disposable
filterwares (Nalge Company, Rochester, NY), and Millex-GS 0.22 pm flter unit (Sigma
Co., St. Louis, MO).
2.2: Methods
2.2.1: Instrumentation
The shaker for ELISA was Bamstead/Thermolyne, Maxi-Mix III™ (Dubyque,
Iowa). Cottonseed product samples were ground in a coffee mill (BRAUN, model
KSM2, Mexico). Mixing usually was performed on the vortex (Scientific Industries,
Inc., Bohemia, NY). Pre-coated thin layer chromatography (TLC) plastic sheets PEI-
Cellulose (MC/B Manufacturing Chemists, Inc., Cincinnati, Oh) were used in
monitoring the reactions of gossypol derivatization. TLC solvent system was the
mixture of ethyl-acetate: 2-propanol: H 2O (65: 24: 11 v/v) and the spots were visualized
under BLAK-Ray Lamp (Model UVL-56, Upland, CA). Cells were grown in a water-
jacketed CO2 incubator (Forma Scientific, Inc., Maijetta, Ohio) and viewed using an
inverted microscope (Nikon, model TMS, Japan). Cell counting was performed on a
Neubauer counting champer (Fisher). Tissue culturing operations were done in a
biological safety cabinet from ENV Service, Inc. (Minneapolis, MN). Clone selection
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from soft agar was performed in a laminar flow hood. All autoclaving was performed at
121°C for 15 min. Matrix-assisted laser desorption/ionization-time of flight-mass
spectrometry (MALDI-TOF-MS) was used to measure the number of gossypol groups
in protein gossypol conjugates.
2.2.2: Modeling and statistical analysis
Enzyme-linked immunosorbent assays (ELISAs) were carried out in 96-well
plates and read at wavelength 405 nm in a SPECTRAmax Plus microtiter plate reader
(Molecular Devices Corporation, Sunnyvale, CA). The software package Softmax
(Molecular Devices) was used for fitting the sigmoidal standard curve based on a four-
parameter logistic method of Rodbard (1981) as an empirical way to describe the dose
response curve in ELISA. The parameter a represents the response at zero concentration
of gossypol. The d is the response at “infinite” concentration of gossypol. The
parameter c is the gossypol concentration giving 50% reduction (halfway between a and
d, called I50 value). The parameter b is the curvature parameter which determines the
steepness of the curve. Those four parameters a, b, c and d are obtained by the method
of least squares of observed data using the software package Softmax (Molecular
Devices). X in the equation is the gossypol concentration, and Y is the corresponding
absorbance unit. After calibration process, the curve is used to determine the
concentrations of the unknowns.
a - d y = ------1 +(x/cf
Graphs were plotted as Log 10 of equivalent gossypol concentration vs. relative
absorbance (A/Ao). Where A is the corresponding absorbance at certain gossypol (or
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. gossypol-derivative) concentration; Ao is absorbance when gossypol (gossypol-
derivative) concentration is 0 .
Four parameter sigmoidal equation was used to calculate I 50 values based on the
least square errors of the observed data. Comparison of I 5 0 values for different gossypol
derivatives were made using Tukey’s Studentized Range Test with significance level of
95%.
2.2.3: Preparation of experimental solutions
Phosphate buffered saline (PBS) solution was prepared by dissolving 18 g NaCl,
2.22 g disodium hydrogen phosphate, and 0.6 g potassium dihydrogen phosphate in 1.9
L of distilled water, and adjusting the pH to 7.3 with IN NaOH, then bringing the
volume to 2.0 L. For phosphate buffered saline-Tween 20 (PBST) solution, Tween 20
(1.0 g) was added into 2 L PBS solution. NH 4 CI solution (0.8% NH 4 CI) was made by
adding 0.8 g of NH 4 CI into 100 ml of distilled water and was autoclaved. Trypan blue
(0.4%) stain solution was prepared by adding 0.4 g of trypan into 100 ml of PBS
solution. ABTS substrate solution was made by adding 10 mg 2,2’-azino-bis(3-
ethylbenthiazoline- 6 -sulfonic acid) (ABTS), 8 pi hydrogen peroxide (30%) solution in
24 ml of 0.1 M citrate buffer (pH 3.8). BSA solution (0.33%) was made by adding
BSA 200 mg into 60 ml of PBS. Dimethylsulfoxide (DMSO) solution ( 8 %) was
prepared by mixing 8 ml of DMSO and 92 ml of sterile fetal calf serum aseptically.
Sterile L-glutamine solution (200 mM) was prepared by adding 3.00g of L-glutamine in
100 ml of PBS and filtered through 0.22 irni of filter kit.
The following media were prepared for the culture of myelomas and
hybridomas. RPMI-1640 medium was prepared by dissolving RPMI 1640 powder
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1.04%) and sodium bicarbonate (0.2%) in distilled water and filtered through 0.45 pm
filter kit in biological safety cabinet. Serum-free RPMI consisted of RPMI 1640
medium with the addition of L-glutamine (2 mM), sodium pyruvate (1 mM) and
penicillin-streptomycin ( 1 0 0 units penicillin and 0 .1 mg streptomycin per ml).
Complete RPMI-20 was made by adding 20% (v/v) calf serum in serum-free RPMI and
complete RPMI-10 was made by adding 10% of calf serum to serum-free RPMI. HT or
HAT medium was complete PRMI-20 with addition of HT (100 p.M sodium
hypoxanthine and 16 pM of thymidine) or HAT (HT with 0.4 pM aminopterin). Spent
medium was prepared by growing myeloma cells in complete RPMI-10 for 2-4 days,
centrifuging the cell suspension at 250 x g for 5 min and passing the supernatant
through 0.45 pm filter kit. Conditioned medium was prepared with spent medium plus
20% fetal calf serum, L-glutamine (2 mM), sodium pyruvate (1 mM) and penicillin-
streptomycin ( 1 0 0 units penicillin and 0 .1 mg streptomycin per ml).
2.2.4: Preparation of Immunogen
A scheme for the production of LPH-G conjugates (Limulus polyphemus
hemolymph-gossypol) via a Schiff base intermediate is shown in Figure 8 . Two LPH-G
conjugates were produced as follows:
LPH-Ga: Gossypol (23.5 mg) was dissolved in 6 ml methanol. LPH (24.5 mg)
was dissolved in 6 ml PBS buffer, then the two solutions were mixed and reacted with
continuous stirring for 48 h at 5°C in the dark. The whole mixture was filtered through a
Whatman No. 1 filter paper and washed using ethyl ether to remove unreacted gossypol.
The product collected on the filter was air dried and stored at 5°C with desiccation.
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD C
*c a o erf u V) Z um as CQ« 2 Crf
JSo i VI
erf I _c 2fS ‘S X oUi a. . . Gossypol-protein conjugate formation via a Schiff base intermediate 8 Figure Figure
o a / to>■» Oc o \ a
40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LPH-G8: Gossypol (24.5 mg) was dissolved in 7 ml methanol and mixed with
24.5 mg LPH dissolved in 25 ml PBS and reacted with continuous stirring for 48 h at
RT under mild nitrogen flow with adding 105.6 mg of sodium cyanoborohydride. After
the reaction, the whole mixture was dialyzed in 1 L of 8 M urea for 24 h, 4 L of 50 mM
ammonium carbonate for 24 h and finally 4 L of 25 mM of ammonium carbonate for 24
h, respectively, then the product was lyophilized.
2.2.5: Preparation of solid-phase conjugates
BSA-G conjugates (Bovine serum albumin-gossypol) were produced via Schiff
bases as follows:
a. Gossypol (7.8 mg) was dissolved in 2 ml of ethanol. BSA (50 mg) was
dissolved in 25 ml PBS. These two solutions were mixed with 90 mg of sodium
cyanoborohydride (NaBHjCN) and reacted for 48 h in the dark under N 2 at RT, then
dialyzed in same way as described for immunogen LPH-G8.
b. Gossypol samples (3.9, 7.8, 15.6 mg) were each dissolved in 2 ml methanol.
Each was mixed with 15 ml of BSA solution (containing 50 mg BSA) plus 60 mg
NaBHaCN. The reaction conditions and purification were performed in same way as
(a).
To observe the effect of reducing agent, controls were also prepared without the
addition of NaBHsCN.
2.2.6: Measurement of gossypol groups in conjugates
The number of gossypol groups in protein gossypol conjugates were measured
using Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry
(MALDI-TOF-MS). All samples were prepared for MALDI analysis by mixing 2,5-
41
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dihydroxybenzoic acid (2,5-DHB) matrix solution with dried BSA-G to give mole ratio
104:1 — 103 :1. The analyte-matrix mixture was applied to the sample probe and was air-
dried. A linear, time lag focusing TOF mass spectrometer was used to collect all mass
spectra at averaging of 290(350) laser shots.
2.2.7: Polyclonal antibody (PAb) production
2.2.7.1: Rabbit immunization
Two female NZW rabbits (#16 and #15) were immunized with 1.0 mg of
conjugate (LPH-GA and LPH-G8, respectively) in 2 ml of PBS/Freund’s complete
adjuvant (1:1, v/v). Rabbit #16 was first immunized at age 6 months and boosts were
given monthly. Rabbit #15 was immunized beginning at age 9 months and boosts were
given on months 10, 15 and 17 using the same concentration of conjugate in Freund’s
incomplete ajuvant. Blood samples were taken 2 weeks after each boost and transferred
to sterile vacutainers, allowed to clot half an hour at room temperature, centrifuged at
16,000 x g for 10 minutes. The sera were tested for anti-gossypol antibodies and stored
frozen. Preimmune sera were used as negative control.
Two weeks after the last immunization, the rabbits were euthanized by injection
with atropine sulfate SA (0.05 ml/kg, sc.) and acepromazine maleate (0.5 ml/kg, sc.).
After 10 min, the rabbits were injected with xylazine (5 ml/kg, sc.) and ketamine (50
mg/kg, sc.), then the rabbits were bled using cardiac puncture and the sera collected and
stored as described above.
2.2.7.2: Antibody capture noncompetitive ELISA for screening sera
An antibody-capture noncompetitive ELISA (ACN-ELISA) was used to monitor
the presence of anti-gossypol antibodies in rabbit sera. BSA-G in PBS (10 pg/ml) was
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prepared and a 100 pi aliquot was added to each well of an Immulon 2 HB microtiter
plate and incubated at 5°C overnight (coating). The solution was removed by inverting
the plate with a quick flick of the wrist, and each well was filled with 2 0 0 pi /well of
1% BSA in PBS (blocking). Following an incubation of 30 min at 37°C, the solution
was removed and wells were washed with 3 x 200 pi of PBST, incubating each wash
for 5 min at RT with shaking. An aliquot of 10% methanol in PBS (50 pl/well) was
added coupled with 50 pl/well of diluted antisera or preimmune sera (1st Ab: diluted
1/1000 with 1% BSA in PBST) and incubated for 30 min at 37°C. After washing away
unbound reagents as described above, the bound antibodies were detected by the
addition of 100 pl/well of 1/10,000 diluted goat anti-rabbit IgG peroxidase conjugated
(2nd Ab) in 1%BSA in PBST, incubated for 30 min at 37°C and then washed with 3 x
200 pi of PBST as described as above. ABTS substrate was added (100 pi /well) and
peroxidase activity was measured as absorbance at 405 run after 30 min of incubation at
RT.
2.2.7.3: Optimization of ELISA
Immulon IB, Immulon 2 HB, immulon 4 HBX and PVC microtiter plates were
compared for their abilities to immobilize solid phase conjugate (coating) in ACN-
ELISA. The blocking buffers (1% BSA, 1% gelatin, 1% ovalbumin and 1% powder
milk), 1st Ab and 2nd Ab dilution buffers (1%, 0.5%, 0.1% or 0.05% BSA in PBST) and
incubation time (30 min, 1 h and 2 h) and temperature (37°C and RT) were investigated
for their effects on ELISA. Acetone and DMF at different concentrations and 10%
methanol incubated with diluted PAb were compared for their effects on ACN-ELISA.
43
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Checkerboard ELISA was used to optimize the concentrations of BSA-G and
antisera from rabbit #16 and #15. A two dimensional titer determination was performed
on the plate with decreasing concentration of coating solution BSA-G rowwise, ranging
from 0.01 pg/ml to 100 pg/ml from top to bottom overnight at 5°C (coating). After
removing the coating solution by inverting the plate, blocking solution 200 n 1/well of
1% BSA in PBS was added. After 1 h incubation at RT (2 h @ RT for rabbit #15 PAb),
the solution was removed and washed with 3 x 200 pi PBST. Fifty pi of 10% methanol
and serially diluted antisera (diluted 1/500 to 1/8000 in 0.5% BSA in PBST, 50 ^1/well)
were added respectively from left to right across columns (non-competitive step).
Following lh incubation at RT (2 h @ RT for rabbit #15 PAb), the unbound materials
were washed away with 3 x 200 |d PBST, and 100 pl/well of 2nd Ab (goat anti-rabbit
IgG peroxidase conjugated) at 1/10,000 dilution in 0.5% BSA was added. After 1 h
incubation at RT (2 h @ RT for rabbit #15 PAb), the excess reagents were removed and
washed with 3 x 200 pi PBST, and 100 pi/ well of ABTS substrate was added. The
absorbance at 405nm was measured after 30 min incubation at RT. The optimum
combination of antiserum and coating solution were chosen both dilution curves were
steep and gave an absorbance between 1 to 2.
2.2.8: Monoclonal antibody (MAb) production
2.2.8.1: Immunization of mice and serum test
Preimmune blood samples (retro-orbital) were taken from five mice (one month
old, female, BALB/c). Mouse #0, #1 and #2 were immunized with 1.0 mg of LPH-GA
conjugate in 2 ml of PBS/Ribi Adjuvant System (1/1, v/v), and mouse #3 and #4 were
immunined with LPH-G8 in the same way. Injections were performed as 0.1 ml
44
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. subcutaneous and 0.1 ml intraperitoneal. Boosts were given in a similar manner on 28,
49, 70 and 91 days after the first immunization. Blood samples were taken 1 week after
each boost and transferred to sterile vacutainers, allowed to clot half an hour at RT,
centrifuged at 16,000 x g for 10 minutes. The sera were collected and tested for anti-
gossypol antibodies using ACN-ELISA.
Microtiter plate was coated (100 pi of 5 pg/ml BSA-G), blocked (200 pi of 1%
BSA) and washed (3 x 200 pi of PBST) as described as above. Fifty pi of 10% of
methanol in PBS and 50 pi diluted serum (diluted 1/1000 in 0.5% BSA in PBST) were
added to microtiter plate. Following 2 h incubation at RT, the unbound material was
removed and washed with 3 x 200 pi in PBST, and 100 pi of anti-mouse IgG+IgM
peroxidase conjugated at 1/3,000 dilution in 0.5% BSA was added. After 2 h
incubation at RT, excess reagent was washed away and 100 pi of ABTS substrate was
added. The absorbance was measured at 405 nm after 30 min incubation at RT.
2.2.8.2: Myeloma cell culture
Myeloma cells (NS-1) were cultured in sterile tissue culture dishes using
complete RPMI-10 at 37°C with 5% CO 2 and at 90-100% relative humidity. Cells were
subcultured every 2-3 days, using 1/5 to 1/20 dilution to keep cell density from 105 to
106 cells/ml. Cells were used for fusion two days after subculturing. After
centrifugation at 250 x g for 5 min, the cells were suspended in 15 ml of RPMI medium,
and centrifuged again. Washing was performed twice, then cells were combined and
resuspended in 2.5 ml of RPMI medium and kept on ice until fusion. Meanwhile, the
cell suspension was counted at 1/10 dilution in 0.25% trypan blue in PBS, using a
Neubauer counting chamber.
45
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.8.3: Recovery of splenocytes
After four immunizations, the mouse #2, exhibiting the highest titer of anti-
gossypol antibodies, was immunized again. Three days later, this mouse was sacrificed
by cervical dislocation, the spleen was aseptically removed, mashed with two single
frosted autoclaved microscope slides, and resuspended in a tissue culture dish
containing 10 ml RPMI medium, and transferred to a centrifuge tube. Cells were
centrifuged at 250 x g for 5 min and washed twice with 10 ml PRMI. After the second
centrifuge, the cells were resuspended in 4 ml of 0.8% NH4CI for 3 min to lyse the red
cells, then centrifuged at 250 x g for 5 min. After discarding the upper-layer, the cells
were resuspended in 7 ml of RPMI medium, counted and immediately used for fusion.
2.2.8.4: Production of hybridomas
Myeloma (10 7) cells were combined with spleen cells (108). The mixture was
mixed on the vortex at low speed, centrifuged at 800 x g for 5 min and the supernatant
was discarded. One ml of 50 % PEG 1500 in HEPES buffer was added to the cells with
gentle mixing over a minute, then mixed on vortex for a few second, and left for another
minute. One ml of RPMI was slowly added and following adding another 9 ml of
RPMI in next 3 min, then the cells were centrifuged. After discarding the supernatant,
the cells were resuspended in 90 ml of HAT medium, and 150 pi of aliquot was added
into each well of 7 sterile 96-well tissue culture plates with doing a few control using
myeloma cells in each plate.
2.2.8.5: Culture, screening, and selection of hybridomas
Fused cells were incubated at 37°C, 5% CO 2 with 90-100% relative humidity.
Seven days after fusion, the cells were fed 100 pi of HT medium per well. Hybridomas
46
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were checked daily after feeding, looking for colonies and yellowing of the medium.
When the medium became yellow and the colonies were visible over 30% of the base of
the individual wells, the supernatants from these wells were screened for Ab production
using ACN-ELISA and ACC-ELISA.
Fifty pi of 10% methanol (for ACN-ELISA) or gossypo 1-derivatives (for ACC-
ELISA) (100 to 250 ng/ml of gossypol equivalents) was incubated with 50 pi of each
supernatant on microtiter plates that were previously coated (100 pi of 5 pg/ml of BSA-
G), blocked (200 pi of 1% BSA) and washed (3 x 200 pi of PBST). The tests were
performed as same as described in section 2.2.8.1 for mouse PAb test.
Cells with supernatants that showing positive in both assays were transferred
into 24-well microtiter plates and fed with about 0.5 ml of complete RPMIR-20 to each
well. After three days, the cells were transferred into 65x10 mm tissue culture dishes
and cultured for 2-4 days, then transferred into 100x20 mm tissue culture dishes.
2.2.8.6: Cell cloning
Once the colonies have been identified as producing anti-gossypol antibodies,
the positive cells were cloned using soft agar and limiting dilution methods.
Soft agar method: A 2.4% agarose medium was prepared by weighing 2.4 g
agarose in 100 ml of 0.15 M NaCl solution, heating to 70°C until dissolved. The clear
solution was distributed into 10 scintillation vials, capped, autoclaved and stored at 5°C
until required.
On the day of cloning, the agarose medium was melt at 37°C, and 2 ml was
transferred into 10 ml of pre-warmed (37°C) conditioned medium to make a 0.48%
47
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. agarose solution (called soft agar). The soft agar was dispensed into 6-polypropylene
tubes with 2 ml for each tube.
One hundred pi of serial uncloned cell dilutions (1/2) in conditioned medium
was added into each of above polypropylene tubes. They were thoroughly mixed on a
vortex mixer and dispensed (0.5 ml /well) into a 24-well microtiter plate with different
dilution for each column. Cells in soft agar were incubated at 37°C with 5 % CO 2 and
90-100% humidity.
After 8-12 days growth, clones were selected from soft agar under an inverted
microscope placed in a laminar flow hood. A mouth-pipeting system was employed by
connecting a sterile 25 pi disposable micropipet, pipet/hose adapter, sterile tubing, two
0.22 pm filter units and a mouth piece (Plhak and Spoms, 1994). Selected cells were
transferred into 200 pi complete RPMI-20 medium in a sterile 96-well tissue culture
plate. They were incubated at 37°C with 5% CO 2 and 90-100% humidity and
subcultured every 2-4 days to keep cell density from 104 to 106 cells/ml (Hum and
Chantler, 1980). Supernatants were screened and positive cells were selected for further
recloning.
Further cloning was performed using a limiting dilution method. Positive cells
were expanded gradually, through 2- and 10-ml culture. Then the cells were diluted
with conditioned medium and plated into 96 well tissue culture plate at 10 cells/well (2
rows), 3 cells/well (3 rows) and 1 cell/well (3 rows). After 9-15 days, the visible
colonies were tested for antibody production. The positive cells were selected from the
highest dilution showing cell growth and cultured.
48
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The cloning procedure was performed three or four times using either soft agar
or limiting dilution methods or both to ensure stability of the cell lines.
2.2.8.7: In vitro production of monoclonal antibodies (MAb)
Antibody was produced by maintaining the hybridomas in complete RPMI-20
and splitting the culture every 2 to 3 days. The supernatant was collected for the assays.
2.2.8.8: Purification of monoclonal antibodies (MAb)
The tissue culture cell suspensions were collected and centrifuged at 250 x g for
5 min to remove the hybridomas. The supernatant from each cell line was transferred
into a beaker and was kept in a cold water bath (0°C). Ammonium sulfate (35 g/100 ml
supernatant) was then slowly (10-15 min) added to obtain 55% saturation with regular
stirring and left to stir for another 30 min. The mixture was centrifuged at 9,000 x g at
2°C for 15 min, and the supernatant was discarded and the protein precipitated was
resuspended in about 2-4 pellet volumes of PBS buffer. This solution was dialyzed
against 3x1500 ml of PBS buffer for 12 h to remove ammonium sulfate. The dialyzed
solution was stored at -20°C.
2.2.8.9: Freezing, storage and thawing of cell line
Healthy cells, approximately 106 to 107 cells, were centrifuged at 250 x g for 5
min, resuspended in 1 ml of 8% dimethylsulfoxide in calf serum and 0.5 ml aliquots
were placed into cryogenic vials. These vials were wrapped in several layers of paper,
and placed into -80°C of freezer for two months, then transferred into a liquid N 2 (-
196°C).
Cells were thawed quickly after removal from liquid tank N 2 in 37°C water until
the ice has just melted. The cells were asceptically transferred into 10 ml of RPMI-20
49
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and mixed thoroughly, then centrifuged at 250 x g for 5 min. Washing was performed
twice to remove DMSO. After the final centrifugation, cells were resuspended in
complete RPMI-20 medium, and cultured.
2.2.9: Crystallization of isomeric gossypol-acetic acid
Crystals of gossypol-acetic acid (Dowd et al., 1998) were formed from acetone
by dissolving gossypol acetic acid into acetone at 1:2.6 (w/w) ratio and crystallization
was performed by storing the solution at -20°C to perform primary nuclei. After
melting most of the precipitated material by mild heating, the crystallization was done
in 4°C.
When crystals grew big enough, they were washed with 10% and 20% acetone
in hexane solutions (v/v) to remove the precipitated gossypol, and each crystal was
placed in a different vial. Isolated crystals were dissolved in acetone, and a small
portion was tested using HPLC after formation of diastereoisomer with (+)- 2-amino-1-
propanol in DMF for 30 min at 95°C. HPLC (Waters 2690, separations module),
performed using a reversed-phase C l8 column (mobile phase: 80% acetonitrile and
20% phosphate buffer pH=3.0) with UV detection at 254 nm, was conducted at USDA,
Southern Regional Research center, New Orleans, LA. After determination, the (+)-
gossypol or (-)-gossypol isomers were combined into 2 separate vials, respectively. The
final products were reanalyzed using HPLC, vacuum dried and stored at 5°C under
desiccation.
2.2.10: Preparation of various forms of gossypol
Gossypol solution and different gossypol derivatives with amino acids and
amine compounds were made as follows:
50
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Derivatizing solutions (0.1M) of L-(+)-Iysine, L-(+)-arginine, ethanolamine, L-
2-amino-l-propanol, 3-amino-1-propanol, para-amino-benzoic acid, L-glutamine, L-(-)-
tryptophan and L-tyrosine were prepared in PBS. Gossypol (2.5 mg/ml) was dissolved
in methanol. Gossypol derivatives were made by mixing 1 part of gossypol solution
and 9 parts of each derivatizing solution, adding 0.3% sodium cyanoborohydride for 2 h
at RT. The reaction was followed by TLC and the absorption spectra of products were
also measured. (+)- and (-)-gossypol isomer derivatives were prepared with L-(+)-
lysine and L-2-amino-l-propanol, respectively, as described above. Gossypol solution
(250 ng/ml) was prepared by diluting 2.5 mg/ml of gossypol in PBS.
2.2.11: Antibody capture competitive ELISA
An antibody capture competitive ELISA (ACC-ELISA) was used for evaluating
the specificity of antibodies to racemic gossypol derivatives, racemic gossypol at
different storage time, and (+)- and (-)-gossypol derivatives.
BSA-G in PBS (5 pg/ml) was coated on the microtiter plate, blocked and
washed as described previously.
For rabbit #15 PAb, 50 pi of serially diluted (1/10) gossypol derivative solutions
with 50 pi diluted serum (diluted 1/1000 with 0.5% BSA in PBST) were added from the
top to bottom row on the plate. The following procedures were same as section 2.2.7.2
for checkerboard titration.
For MAb, 50 pi of serially diluted gossypol derivative solutions (1/2.5 dilution
for 7G2F2G3C6, 1/10 dilution for 6G7D11F10F2F8 and 6G7D11F10F2H3) with MAb
supernatant (50 pi) were added and incubated for 2 h at RT. The following steps
followed section 2.2.8.1 for mouse serum test.
51
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Gossypol solution freshly made and the solution stored in refrigerator for 21
days were compared in ACC-ELISA as competitors for MAb (6G7D11F10F2F8 and
6G7D11F10F2H3) and PAb.
2.2.12: Gossypol analysis in cottonseed products
Cottonseed product samples were ground in a coffee mill for 40 seconds.
During grinding, the start button was released 3 times, each time for 3 seconds.
Method A:
Sample preparation:
For Free Gossypol (FG) analysis, 0.5 g ground sample was accurately weighed
into a 250 ml screw-capped volumetric flask, which had a glass bead-covered bottom.
Fifty ml of 70 % aqueous acetone was added, and the flask was mechanically shaken for
1 h. The slurry was filtered through Whatman No. 2 filter paper. The filtrate was
purged under mild nitrogen flow for 5 h to remove the acetone. Gossypol derivatization
was performed by adding 5 ml of methanol and 219 mg of L-(+)-lysine with NaBHsCN
and bringing the volume up to 50 ml with 0.1 M L-(+)-lysine. The mixture was
mechanically shaken for 2 h at RT.
For Total Gossypol (TG) analysis, a ground sample (about 0.5g) was accurately
weighted into a 50 ml stopered glass bottle. Two ml of methanol and 18 ml of 0.1 M L-
(+)-lysine with NaBHaCN were added and the bottle was vigorously shaken for 2 h at
RT.
Standard solution: Gossypol-lysine derivative was prepared by mixing L-(+)-
lysine and gossypol solution with addition of sodium cyanoborohydride (section
2.2.10).
52
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For the determination of gossypol, an ACC-ELISA was employed using
Immulon 2 HB plates. Standards (gossypol-lysine complex) and samples were analyzed
together on the same plate. Fifty pi of gossypol-lysine (in series of dilutions from 250
pg/ml to 0.001 pg/ml in 10% methanol) or prepared samples (1/10 and 1/100 dilutions
diluted using 10% methanol) with 50 pi of diluted rabbit #15 PAb (1/1000) were added
into pre-coated (5 pg/ml of BSA-G) microtiter plates. The following procedures were
same as section 2.2.11.
Method B:
Free Gossypol (FG) analysis: FG was extracted from ground samples (about
0.5g) using 70% aqueous acetone and filtered through Whatman No. 2 filter paper and
the first 5 ml of filtrate was discarded (AOCS, Ba 7-58). Three ml of the remaining
filtrate was transferred into a 20 ml scintillation vial, and 18 ml of 0.12 M of L-(+)-
lysine with NaBHsCN was added. The reaction was performed at RT for 2 h with
vigorous shaking.
Standard solution: 32.6 mg of gossypol-acetic acid was treated the same as
described above.
For the determination of gossypol, an ACC-ELISA was employed using
Immulon 2 HB plates. Fifty pi of gossypol-lysine standard solutions (in series of
dilutions from 250 pg/ml to 0.001 pg/ml) or prepared samples (1/10 or 1/100 dilutions
diluted using 10% acetone) with 50 pi of diluted PAb (1/1000) were added into pre
coated (5 pg/ml of BSA-G) microtiter plates. The following procedures were same as
section 2.2.11.
53
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3
RESULTS AND DISCUSSION
3.1: Effects of reducing agent and pH on production of protein-gossypol conjugates
Molecules below 5,000 daltons usually are not effective immunogens
(Crowther, 1995a). Gossypol (MW 518) is not large enough to elicit a significant
immune response, therefore it must be conjugated to a carrier protein to be used as an
immunogen. The carrier protein must be of a high molecular weight (typically greater
than 20,000), and phylogenically unrelated to the animal species in which the antisera
are to be raised (Jenner and Law, 1996). For anti-gossypol antibody production, LPH
(Limulus Polyphemus hemolymph), a hemolymph protein (high MW) from horseshoe
crab was selected as a carrier protein.
In order to immobilize gossypol on the surface of microtiter plates, gossypol
was bound to another protein BSA (bovine serum albumin) to be used as immobilized
antigen (Ag) during immunoassay. BSA contains 59 lysines with 30 to 35 of these
available for derivatization with aldehydic groups (Hermanson, 1995).
Gossypol (carbonyl compounds) react very readily and reversibly with amino
groups in proteins to form Schiff bases. The reaction is pH-dependent with the greatest
reaction efficiency at pH 9-10 (Hermanson, 1995; Conkerton and Frampton, 1959).
Gossypol, however, can be rapidly attacked in alkaline solution by atmospheric oxygen
(Scheiffele and Shirley, 1964). The pH of the reaction was therefore controlled around
pH 7.3 and the products formed were stabilized with sodium cyanoborohydride, which
can convert the Schiff base to a more stable secondary amine (Hermanson, 1995).
54
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MALDI-TOF-MS (Matrix-assisted laser desorption ionization-time of flight-
mass spectrometry) provides a great versatility in MS analyses by allowing formation of
large ionic species from thermally labile compounds. Its impressive mass range could
surpass 106 daltons (Spoms and Abell, 1996). Our results showed that LPH-G
conjugates could not be detected by using MALDI, probably because of the extremely
large aggregate (MW3,000,000) that LPH forms (Szurdoki et al., 1995). MALDI-TOF-
MS results (Figure 9, Figure 10 and Figure 11) for BSA-G indicated that the reaction
was favored in the presence of reducing agent NaBHjCN with the ratio of
gossypol/BSA in conjugates 2 times higher than that without NaB^CN (Table 2).
Table 2. Gossypol groups per BSA in BSA-G conjugates
Mole ratio used in reaction Gossypol/BSA in conjugate * (Gossypol/BSA) With NaBH3CN Without NaBH3CN
42/1 11.5 -
20/1 10.8 6.14
10/1 9.5 4.5
20/1 (ethanol) 6 3 ♦Apparent average molecular weights of BSA (bovine serum albumin) and BSA-G (bovine serum albumin-gossypol) conjugates were determined using MALDI-TOF-MS (Matrix-assisted laser desorption ionization-time of flight-mass spectrometry), and gossypol groups per BSA were determined by calculation.
In addition to confirming the production and stabilization of our gossypol-
protein conjugates, we may extrapolate these findings to ruminant vs. nonruminant
metabolism. The stabilization of gossypol-protein complexes by the reducing agent
NaBHsCN could explain why ruminant animals are less susceptible to gossypol
intoxication than monogastric animals. The rumen is a highly reducing environment,
55
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced
Counts 1000 -time of flight-mass spectrometry) spectrograms of BSA (bovine serum serum (bovine BSA of spectrograms spectrometry) flight-mass of -time Figure 9. MALDI-TOF-MS (Matrix-assisted laser desorption ionization ionization desorption laser (Matrix-assisted MALDI-TOF-MS 9. Figure albumin) and BSA-G (bovine serum albumin-gossypol) conjugates albumin-gossypol) serum (bovine BSA-G and albumin) BSA-G/NaBH3CN(10/l mole ratio for reaction) for ratio mole BSA-G/NaBH3CN(10/l BSA-G (10/1 mole ratio for reaction) for ratio mole (10/1 BSA-G 3000 Mass(m/z) 56 007000 5000 68776.5 1356.7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced
Counts 1000 Figure 10. MALDI-TOF-MS (Matrix-assisted laser desorption ionization ionization desorption laser (Matrix-assisted 10.MALDI-TOF-MS Figure -time of flight-mass spectrometry) spectrograms of BSA (bovine serum serum (bovine BSA of spectrograms spectrometry) flight-mass of -time albumin) and BSA-G (bovine serum albumin-gossypol) conjugates albumin-gossypol) serum (bovine BSA-G and albumin) BSA-G/NaBH3CN(20/l mole ratio for reaction) for ratio mole BSA-G/NaBH3CN(20/l BSA-G (20/1 mole ratio for reactio for ratio mole (20/1 BSA-G 3000 Mass(m/z) 57 5000 7000 66431 69615.6 72052 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced
Counts 00 00 00 7000 5000 3000 1000 Figure 11. MALDI-TOF-MS (Matrix-assisted laser desorption ionization ionization desorption laser (Matrix-assisted MALDI-TOF-MS 11. Figure BSA-G/NaBH3CN (20/1 mole ratio for reaction in ethanol in reaction for ratio mole (20/1 BSA-G/NaBH3CN -time of flight-mass spectrometry) spectrograms of BSA (bovine serum serum (bovine BSA of spectrograms spectrometry) flight-mass of -time albumin) and BSA-G (bovine serum albumin-gossypol) conjugates albumin-gossypol) serum (bovine BSA-G and albumin) BSA-G (20/1 mole ratio for reaction in ethanol) ethanol) in reaction for ratio mole (20/1 BSA-G Mass(m/z) 58 66431 | 3 69459.4 | having a redox potential of -0.4 volts (Brock et al., 1994) with a high microbial protein
supply (Van Soest, 1982). High protein content increases the chances for binding free
gossypol and the reducing environment favors stabilization of protein-gossypol Schiff
base intermediates. Moreover, gossypol contains two types of potentially reactive
functional groups, aldehydes and phenols. The condensation of gossypol with other
phenols and formation of insoluble metal complexes may be also involved in gossypol
detoxification in ruminants (Muzaffaruddin and Saxena, 1966; Ramaswamy and
O’Connor, 1968).
3.2: Production of polyclonal antibodies (PAb)
3.2.1: Monitoring PAb production
Figure 12 and Figure 13 show the increase in antibody over the immunization
period, tested using an antibody-capture noncompetitive ELISA (ACN-ELISA). The
increase reflected either a greater concentration or a higher binding affinity of anti-
gossypol antibodies in the rabbit serum over time. Both sera showed the highest titers
after four immunizations.
3.2.2: Optimization of ELISA for PAb
Several parameters, including microtiter plates, blocking buffers, 1st Ab and 2nd
Ab dilution buffers, incubation time and temperature, acetone and N,N-
dimethylformamide solvents, concentrations of coating solution (BSA-G) and antisera,
were investigated for their effects on ELISA.
3.2.2.1: Effects of microtiter plates on immobilization of BSA-G
The first step of an indirect ELISA is to directly coat BSA-G on the plastic plate
surfaces by passive adsorption. The rate of coating BSA-G to plates depends on the
59
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10.5 9.5 Age (months) Arrows represent immunizations and squares represent bleeds. 6.5 7.5 8.5 Figure Antibody 12. production during immunization period from rabbit #16. 6 0.4 0.4 - 0.6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Age (months) Arrows represent immunizations and squares represent bleeds. Figure 13. Antibody production during immunization period from rabbit #15 9 9 9.5 10.5 15.5 17.5 0 1.4 1.2 0.8 0.6 0.4 0.2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. temperature, the BSA-G concentration and the type of solution. The higher
temperature, the greater the rate of adsorption (Crowther, 1995b). The most usual
regimens involve incubating at 4°C overnight or at RT for 1-3 hours. Considering the
instability of gossypol, increasing temperature may have a deleterious effect on the
antigen gossypol, so we selected 4°C overnight for the coating step. Under this
condition, different microtiter plates were compared including flexible
polyvinylchloride (PVC) and treated plates, such as Immulon 1 B, Immulon 2 HB and
Immulon 4 HBX. Of these plates, BSA-G produced different signal intensities
depending on the plates used (Figure 14 and Figure 15). Immulon 2 HB and 4 HBX
were found to have the highest signal to noise ratio (absorbance when 10 |ig/ml of
BSA-G used divided by absorbance when 0 pg/ml of BSA-G used) when compared to
Immulon 1 B or flexible PVC plates. According to the manufacturer (Dynex
Technologies Inc.), Immulon 2 HB was irradiated to provide high binding affinity for
proteins primary with hydrophilic tendencies and Immulon 4 HBX was treated to
improve protein adsorption in a hydrophilic environment. Immulon 1 B is suggested for
adsorption of proteins in a hydrophobic environment. In this study, BSA-G was
dissolved in PBS, so the greater adsorption on Immulon 2 HB or Immulon 4 HBX is
reasonable.
3.2.2.2: Selection of assay buffers
There are a wide variety of buffers used in ELISA, including those for coating,
blocking, primary and secondary antibody dilutions, washing and peroxidase substrate.
The solutions used for each stage of the ELISA are important for successful assay
development.
62
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 [BSA-G], pg/ml 0.1 1B (3.43) 1B Immulon 2HB (7.69) 2HB Immulon 0.01 (1.36) po|yvinylchloride Flexible — Immulon 0 The numbers shown in parentheses are signal to noise ratios (Abs.@ pg/ml/ 10 Abs.@ 0 pg/ml). 0.5 0.6 0.4 0.3 0.2 were coated with BSA-G (bovine serum albumin-gossypol) solutions and serum was diluted 1/1000. Figure 14. Comparison ofmicrotiter plates on ELISA for rabbit #16 PAb (polyclonal antibodies). Plates
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 0.1 [BSA-G], |ig/ml 0.01 ■*— Flexible polyvinylcliloride (4.03) polyvinylcliloride Flexible ■*— IB (1.76) -♦—Immulon 2HB (5.34) Immulon ■*— 4HBX (5.18) Immulon *— 0 The numbers shown in parentheses are signal to noise ratios (Abs.@ 10 pg/ml/ Abs.@ 0 pg/ml). 0.4 . 0.2 were coated with BSA-G (bovine serum albumin-gossypol) solutions and serum was diluted 1/1000. Figure 15. Comparison ofmicrotiter plates on ELISA for rabbit #15 PAb (polyclonal antibodies). Plates cn o < X> n O
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. High background can be caused by the nonspecific interaction of assay
components with the solid-phase. Nonspecific adsorption of protein can take place with
any available plastic sites not occupied by solid-phase reagent or by non-specific
binding to other protein reagents used in the assay. Blocking reagents were applied to
minimize this problem. They act by competing with nonspecific factors for available
sites. The criterion to choose a good blocker is to yield the highest signal to noise ratio.
Four blocking proteins: gelatin, casein, BSA and powdered milk, were compared for
their effects on signal to noise ratios. The results (Figure 16) showed that BSA and
milk gave higher signal to noise ratios than either casein or gelatin at 1% concentration.
Milk powder is much cheaper than BSA, and it was recommended as a promising
candidate for blocking by Vogt (1987). However, it must be used with care in ELISA
systems, because it is a complex mixture containing substances that may interfere with
assay reagents (Vogt, 1987). BSA is relatively pure, well characterized and readily
soluble, making it valuable as a blocking reagent. In this study, BSA was chosen as
blocking protein for these reasons.
Primary and secondary antibodies in dilution buffers containing 1, 0.5, 0.1 or
0.05% BSA in PBST were compared in ACN-ELISA. The results (Figure 17 and 18)
showed that high BSA concentration in PBST is more efficient at reducing the non
specific binding than lower BSA concentrations, especially for rabbit #16 PAb where
non-specific binding seemed to be a more serious problem. BSA in buffer could
compete with 1st and 2nd Ab for nonspecific binding sites on the immobilized phase.
Higher BSA concentrations gave lower background absorbance and also suppressed the
binding of 1st Ab and/or 2nd Ab to immobilized phase.
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [BSA-G], pg/ml 0.1 10 BSA (9.0) milk (7.7) gelatin (5.8) ovalbumin (7.1) 0.01 0 5 0 1.5 0.5 1/1000. 1/1000. The numbers shown in parentheses are signal to noise ratios (Abs.@ pg/ml/ 10 Abs.@ 0 pg/ml). albumin. Plates were coated with BSA-G (bovine serum albumin-gossypol) solutions and serum was diluted c/) < o ■'3- X) Figure 16. Comparison ofblockers on ELISA for rabbit #15 PAb (polyclonal antibodies). BSA is bovine serum ON 0\
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 [BSA-G], pg/ml 0.1 1%BSA (4.65) 1%BSA 0.5%BSA (4.01) 0.5%BSA (2.23) 0.1%BSA 0.01 0 0 1.5 0.5 1/1000. 1/1000. The numbers shown in parentheses are signal to noise ratios (Abs.@ pg/ml/ 10 Abs.@ 0 pg/ml). (A < o IT> <§> X> albumin. Plates were coated with BSA-G (bovine serum albumin-gossypol) solutions and serum was diluted Figure 17. Effect ofdilution bufferon ELISA forrabbit #16 PAb (polyclonal antibodies). BSA is bovine serum CT\
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 [BSA-G], |ig/ml 0.1 1% BSA(12) 1% 0.5% BSA (12.5) (12.5) BSA 0.5% 0.1% BSA (8.2) (8.2) BSA 0.1% (10.8) BSA 0.05% 0.01 0 2 0 1.5 0.5 1/1000. The numbers shown in parentheses are signal to noise ratios (Abs.@ 10 pg/ml/ Abs.@ 0 pg/ml). albumin. Plates were coated with BSA-G (bovine serum albumin-gossypol) solutions and serum was diluted Figure 18. Effect ofdilution buffer on ELISA for rabbit #15 PAb (polyclonal antibodies). BSA is bovine serum (A < O n O oo
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Detergents are included in washing steps and in 1st and 2nd Ab dilution buffers,
where they act to reduce non-specific binding. They reduce the surface tension of the
incubation media, thus inhibit hydrophobic interactions between proteins (1st Ab with
blocker, 2nd Ab with blocker), between proteins and sample, and between the reagents
and the uncoated surface. Detergent concentration should be kept as low as possible to
minimize the interference with Ab-Ag binding. Tween 20 at 0.5%, 0.1% and 0.05%
were compared in ELISA and they all efficiently decrease the nonspecific binding, so
the lowest concentration of 0.05% was selected.
3.2.2.3: Effects of incubation time and temperature on interactions of Ab-Ag
Incubation time and temperature are two important parameters for ELISA. The
interactions of Ab-Ag are noncovalent and reversible. Ab-Ag reactions in solid-liquid
interface are limited by mass transport or steric interactions (Stenberg and Nygren,
1988). The overall reaction scheme can be expressed as
ka Ag + Ab ** Ab-Ag kd
Where ka is the effective forward association constant and kd is the effective reverse
dissociation constant. The temperature can influence both reaction rate constants (ka
and kd), and the effect is not same for both. Kemeny (1991) found that near maximal
binding could be achieved within 2 hours for most antigens and antibodies. In the
current study, the incubation times (1 h vs. 2 h) and temperature (RT vs. 37°C) for both
of the antibody incubation steps (1st and 2nd antibodies) of ELISA were compared
(Figure 19 and Figure 20). Incubation for 2 h at RT gave greater signal to noise ratios
than either 30 min at 37°C or 1 h at RT. It is important that incubation time is long
69
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 1 [BSA-G], pg/ml 0.1 25°C (a> lh@ 25°C 30min 37°C(5) 2h 0.01 albumin-gossypol) solutions and serum was diluted 1/1000. PAb (polyclonal antibodies). Plates were coated with BSA-G (bovine serum 0 3 0 2 Figure 19. Effect ofincubation time and temperature on Ab-Ag interactions for rabbit #16 1.5 2.5 0.5 c /i < o o
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 [BSA-G],|ig/ml 0.1 25°C 30min (3) 37°C (3) 30min 0.01 albumin-gossypol) solutions and serum was diluted 1/1000. PAb (polyclonal antibodies). Plates were coated with BSA-G (bovine serum 0 3 2 0 .5 Figure 20. Effect ofincubation time and temperature on Ab-Ag interactions forrabbit #15 2.5 0.5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. enough for Ab-Ag binding, so that the reaction is at or near equilibrium. After this
point, increased incubation time would not significantly affect the amount of colored
product obtained. Nevertheless, time can be shortened at the expense of signal, if a
faster analysis is desired but this would require further testing.
3.2.2.4: Effects of solvents on antibody capture noncompetitive ELISA
Ultimately this assay would be used to measure gossypol in cottonseed products.
For the preparation of gossypol sample and standard, certain solvents must be used for
dissolving gossypol or rupturing glands and this solvent must be compatible with
ELISA. Current available methods for gossypol analysis use either acetone or DMF for
sample preparation. These solvents and methanol were chosen to incubate with the 1st
Ab in an antibody capture noncompetitive ELISA format to predict whether these
solvents could be used in competitive ELISA for sample delivery. As acetone and
DMF concentration increased, serum reactivity was inhibited (Figure 21 and Figure 22).
Acetone concentrations equal to or greater than 10% reduced the reactivity of serum by
46%; DMF concentration, even at 4%, reduced the reactivity of serum by 50%; and
10% methanol gave similar signal as water at 10 pg/ml of BSA-G as coating solution.
Similar effects on the immunoreactivity of antibodies have been reported for organic
solvents (Chen et al., 1995). During the incubation of solvents and antibodies, acetone
and DMF may denature some Ab (proteins) or their binding sites, decreasing the
effective antibody concentration. This could result in lower signals. Therefore,
samples or standard solutions containing acetone or DMF solvents must be diluted in
ELISA to an acceptable range. Further investigation on the effects of these solvents on
the competition ELISA is necessary.
72
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3U0J30V %0t>
suopoy %oe
suopoy o/0Q 3
ouopoy %o I
auopoy %g
3uopoyo/0g'3
suopoy %g3' I
HQ3IAI%0I
OzH Figure Figure 21. Effect of acetone concentration on ACN-ELISA (antibody capture noncompetitive ELISA)
uiu sot1 ® sqy
73
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (antibody (antibody capture noncompetitive ELISA) Figure Figure 22. Effect of N,N-dimethylformamide concentration on ACN-ELISA
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.2.5: Effects of Ab and BSA-G concentrations
The optimal concentrations of Ab and solid-phase conjugates were determined
by checkerboard ELISA (Figure 23 and Figure 24). Increasing the coating antigen,
BSA-G, above 5 pg/ml did not significantly increase the overall absorbance. Therefore,
the optimum concentration of coating antigen conjugate was determined to be 5 pg/ml.
The optimum combination of antiserum and coating solution were chosen where they
gave steep dilution curves and an absorbance between 1 to 2. Serum dilutions of 1/1000
were used in the remaining studies presented in this dissertation.
3.2.3: Effects of immunogen preparation method on PAb production
The results from serum screening and ELISA optimization indicated that sera
from different rabbits showed different behaviors. One reason could be due to animal
to animal variation in immune response. Other reasons may be the chemistry used for
formation of immunogen and immunogen purity (Skerritt, 1995).
The chemistry of immunogen formation plays an important role in determining
assay sensitivity and assay specificity. Careful choice of conjugation to the carrier
protein is necessary (Harrison et al., 1991a and 1991b). The presence of spacer arms
could maximize the exposure of the analyte for antibody production and increase the
assay sensitivity (Chen et al., 1995). In this research, gossypol was bound to e-amino
groups of lysine on the protein. The hapten design and synthesis are critical steps to
elicit a good immune response. Rabbit #16 was injected with LPH-GA without
stabilization, and rabbit #15 was injected with LPH-G8 with the addition of NaBfyCN
for product stabilization. Though substitution ratios could not be detected using
MALDI because of the aggregation tendency of LPH, it can be deduced from the results
75
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 5 [BSA-G], pg/ml 0.1 1/500 1/2000 1/4000 1/8000 ^ 0.01 0 3 2 0 1.5 2.5 0.5 Figure 23. ELISA checkerboard for rabbit #16 PAb (polyclonal antibodies). Plates were coated with BSA-G (bovine serum albumin-gossypol) solutions. Different lines represent different PAb dilutions. i/i < ■'T in o X> ON
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 10 5 [BSA-G], ng/ml 0.1 1/500 1/4000 1/8000 1/2000 -o- 1/1000 0.01 0 0 3 9 1.5 0.5 2.5 Figure 24. ELISA checkerboard for rabbit #15 PAb (polyclonal antibodies). Plates were coated with BSA-G (bovine serum albumin-gossypol) solutions. Different lines represent different PAb dilutions. C /3 < Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of BSA-G conjugation that LPH-G8 with stabilization might also have a greater number of gossypol groups per LPH than LPH-GA. Also, the secondary amino produced by stabilization would be expected to provide a more flexible linking arm, perhaps making the hapten more accessible. The incorporation ratio between hapten and carrier protein LPH is important to get a good immune response. The data from Erlanger (1980) suggested that incorporation levels on BSA from 8:1 to 25:1 gave a good response. For different sizes of carrier proteins, different molar substitution ratios may be required. The packing density is important in the hapten-protein conjugate. Thus, a large carrier protein should have a greater molar substitution ratio than small proteins to maintain the same packing density (Jenner and Law, 1996). The substitution ratio may be one reason to cause the different immune response in this research. Purification of the crude immunogen to remove non-covalently bound haptens may have also affected the Ab response. Haptens may be adsorbed to the carrier in a way which presents an epitope differently than that of the chemically conjugated material. Injecting an immunogen containing both covalently linked and adsorbed hapten could result in the generation of a mixed population of antibodies with different specificities (Jenner and Law, 1996). LPH-GA and LPH-G8 were purified using different methods which may have given different immunogenic properties. 3.3: Production of monoclonal antibodies 3.3.1: Mouse immunization After mice were immunized four times, all five mice showed production of anti- gossypol antibodies by ACN-ELISA (Figure 25). Mouse #2 showed the highest titer of antibody and this mouse was selected as the spleen cell donor to fuse with myeloma. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure Figure 25. Antibody production from mice after four immunizations sot? ® ‘sqv 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.3.2: Production and selection of hybridomas Spleen cells (108) were fused with myeloma (107) using sterile PEG-1500 fusion solution. The fused cells were suspended in HAT selection system to grow the hybridoma and prevent growth of unfused myeloma. After fusion, the plates were examined daily for contamination, pH (by color of medium) and colony growth. After 10-15 days, the cells showing higher signals (72 wells) than control (myeloma) in ACN-ELISA were transferred into 24-well plates and cultured in RPMI- 20 medium. After three days, supernatants from wells (1A7, 1C6, 1F9, 2G5, 3A5, 3D 10, 4C7, 4D8, 4F2, 5D2, 5C5, 5D7, 3H9, 5H9, 6B10, 6G7, 6H2, 7G2) still showing positive in ACN-ELISA were tested using ACN- and ACC-ELISA simultaneously. Cells showing both high absorbance (Ao) in ACN-ELISA and low absorbance (A) in ACC-ELISA with 100 pg/ml of gossypol-lysine are shown in Table 3. At this stage, healthy and positive cells from the wells 1C6, 3H9, 5D7, 5H9, 6G7, 6H2 and 7G2 were selected for cloning. Table 3. Competition using gossypol-lysine (100 pg/ml of gossypol equivalents) as competitor. A is the absorbance from ACC-ELISA and Ao is the absorbance in ACN- ELISA. Cell line A/Ao Cell line A/Ao 1A7 0.3695/1.006 5D2 0.5652/2.9805 1C6 0.455/0.819 5C5 0.2817/0.4472 1F9 0.3437/1.7702 5D7 0.623/1.557 3A5 0.3875/0.6945 6G7 0.441/0.797 3D10 0.2833/0.3808 6H2 0.295/0.5050 4C7 0.5126/1.199 7G2 0.655/1.066 4F2 0.317/0.971 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The semi-solid agar method was used to clone cells 6G7. Eight days after cloning, the colonies were picked from soft agar and transferred into 200 pi of RPMI-20 medium in 96-well plate. After culturing and propagation, the cells from 6G7D11, 6G7D2, 6G7D7, 6G7G3, 6G7B6, 6G7D12 showed positive in ACC-ELISA using lysine-gossypol, arginine-gossypol, ethanolamine-gossypol and 3-amino-1-propanol- gossypol as competitors (Appendix I). 6G7D11 was selected and recloned using the limiting dilution method. Limiting dilution was also used to clone cells from wells 1C6, 3H9, 5D7, 5H9, 6H2 and 7G2. Twelve to fifteen days after cloning, the cells from the highest dilution were tested using ACN- and ACC-ELISA, and the positive ones (5D7D3, 5D7E3, 5D7F3, 5D7E4, 5D7F4, 5D7G4, 7G2A5, 7G2B5, 7G2A6, 5H9F8, 5H9F2) (Appendix I) were recloned using limiting dilution. Poisson distribution statistics indicates that if the most probable cell number per well is 1, then 37% of wells will have no colonies at all, and it is highly probable that the cells growing within each of the individual wells are derived from a single parent cell (Zola, 1987a). After three clonings, 7G2F2G3C6 was obtained, and both 6G7D11F10F2F8 and 6G7D11F10F2H3 were obtained from the thrice cloned cell line 6G7D11F10F2. 3.3.3: Correlation between cell growth and antibody production Once stable hybrid clones secreting anti-gossypol antibodies were obtained, a batch MAb was produced and semipurified. The three cell lines were maintained in RPMI-20 medium and subcutured to keep the cell density within the range of 105-106 cells/ml. Cell growth rate and Ab production of cell line 6G7D11F10F2F8 were monitored. Cell numbers doubled about every 24 h, and when the cell number reached 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4xl06 cells/ml, cells began to die and antibody concentration decreased (Figure 26 and Figure 27). In order to maximize antibody yield from culture, the supernatants should therefore be collected when cells are at peak of cell density or at around 3 days for the conditions employed here. The collected supernatants were combined and concentrated using 55% saturated ammonium sulfate, dialyzed, and stored in a -20°C freezer. 3.4: Effects of reaction pH and structure of derivatizing reagents on gossypol derivatization Gossypol derivatives with L-(+)-lysine, L-(+)-arginine, ethanolamine, L-2- amino-1 -propanol, 3-amino-1-propanol, para-amino-benzoic acid, L-glutamine, L-(-)- tryptophan and L-tyrosine were made in PBS buffer solution (pH7.3). At their isoelectric point (pi), amino acids exist as zwitterions (Lehninger et al., 1992). Gossypol is a weakly acidic compound because of its phenol groups. At pH7.3, both gossypol and the carboxyl group of an amino acid (pl<3) would bear negative charges and repel each other. This would slow down the reaction between these compounds. Therefore, an amino group far from the carboxyl group, such as the e-amino group of L- (+)-lysine and the amino groups of L-(+)-arginine would be expected to react faster than an amino group near to the carboxyl group, such as the a-amino groups of L-glutamine, L-(-)-tryptophan and L-tyrosine. TLC results showed that gossypol disappeared after 10 min at RT in the reaction with L-(+)-lysine and L-(+)-arginine, and the formation of a gossypol complex resulted in the maximum absorption shifting from 386 nm (for unreacted gossypol) to 404-406 nm for gossypol-lysine and gossypol-arginine (Figure 28). After 2 h at RT, TLC showed that some gossypol was still underivatized in the 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o 00 Tl“ o Figure Figure 26. The growth rate of MAb cell line (monoclonal antibody cell line: 6G7D11F10F2F8) , 0 1 x-ou [P3 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. oo Figure Figure 27. MAb (monoclonal antibody cell line: 6G7D11F10F2F8) production during culture urc SOt? © ‘sqv 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. •5 obr* v CO> a. a. Gossypol b. L-(+)-lysine-gossypoI c. L-(+)-arginine-gossypol Figure Figure 28. Absorption spectra of gossypol and gossypol derivatives in methanol, 10% CO 00 CO CM o soircqjosqv 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reactions of gossypol with L-glutamine, L-(-)-tryptophan, L-tyrosine and para-amino- benzoic acid under the same conditions. 3.5: Isolation of (+/-)-gossypol isomers In order to evaluate the antibody specificity for (+)- and (-)-gossypol isomers, isomeric gossypol was isolated through gossypol crystallization from acetone. Each individual crystal contains only one isomer (Dowd, 1998). This unique crystallization property of gossypol makes it valuable for the isolation of extremely pure (+/-)-isomers. Isolated isomers were identified using HPLC (Figure 29). The results showed that the purity for combined (+)-gossypol crystals was 99.96% and for combined (-)-gossypol was 99.64% (Appendix II for area count). Slight contamination (<0.04% for (+)- gosypol and <0.4% for (-)-gossypol) probably resulted from contamination of the tweezers used to pick up the crystals. 3.6: Evaluation of antibody specificity using different forms of gossypol in an antibody capture competitive ELISA It has been known that antibodies produced against a given immunogen will recognize the compound’s different moieties to different degrees. Racemic gossypol derivatives, (+)- and (-)-isomeric gossypol derivatives and gossypol (section 2 .2 . 1 0 ) as standard solutions were used in an antibody capture competitive ELISA (ACC-ELISA) to evaluate the specificities of the PAb and MAb. Four parameter sigmoidal equations were used to determine I 50 values based on the least square errors of the observed data. For the purpose of discussion, the chemical structure of gossypol can be divided into three recognizable moieties. These include 1) a derivatized naphthyl group, 2) a naphthyl group without derivatization, and 3) the inter naphthyl bond. For PAb from rabbit #15, about 0.2 pg/ml of gossypol equivalents caused 50% inhibition of binding 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. v) sa co 3.50 4.00 3.50 4.00 CO CO o CM co CM 2.50 3.00 CD 2.50 3.00 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (I50) when gossypol derivatized with 3-amino-1-propanol, 2-amino-1-propanol, ethanolamine, L-(+)-lysine, and L-(+)-arginine were used as competitors. These results indicate that this PAb could not recognize the difference between these four gossypol derivatives (I5 0 values were not significantly different in Tukey’s Studentized Range Test with significance level of 95%), possibly because PAb may only recognize a naphthyl group on gossypol without derivatization. Gossypol and gossypol derivatives with para-amino-benzoic acid, L-glutamine, L-(-)-tryptophan and L-tyrosine as competitors, however, gave much higher I50 values (Figure 30 and Figure 31). The Ab may not recognize free gossypol, since it was raised to bound gossypol during immunization, no competition between free gossypol and immobilized gossypol. It may also be feasible that underivatized gossypol (in gossypol solution and gossypol derivative reaction solutions) covalently binds to antibodies through Schiff base during the ELISA. This would reduce the effective concentration of the competitor. The covalently bound antibodies with gossypol still could interact with immobilized antigens on Ab-Ag binding sites through hydrogen bonds, hydrophobic forces, Van Der Waals forces and electrostatic forces. These all would decrease the effective competition between free antigens and immobilized antigens, increasing the I50 values. Figure 32, Figure 33 and Figure 34 show the inhibition curves for gossypol derivatives using three different MAb. MAb from different cell lines had different affinities/specificities to different gossypol derivatives. When gossypol derivatives were used as competitors, I50 values ranged from 20 to 55 |ig/ml for 7G2F2G3C6, from 8 to 26 pg/ml for 6G7D11F10F2F8, and from 7 to 24 pg/ml for 6G7D11F10F2H3 (Appendix III). The results indicated that MAb with different specificities have 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 250 2.5 [Gossypol equivalents], pg/ml 0.00025 0.0025 0.025 0.25 are I50 values (theare concentrationsI50 which caused 50% inhibition ofbinding). lysine-gossypol (0.197)lysine-gossypol arginine-gossypol arginine-gossypol (0.209) -propanol-gossypol 3-amino-1 (0.174) 2-amino-1 -propanol-gossypol 2-amino-1 (0.195) gossypol (underivatized) (>250) —ethanolamine-gossypol (0.225) 0 0 0.8 0.6 0.2 0.4 (polyclonal antibodies). Each point is the average of 2 determinations. The numbers shown in parentheses Figure 30. ELISA inhibition curves using gossypol and different gossypol derivatives for rabbit #15 PAb csi t/j <1 o < X X 00 vo Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12500.000125 0.00125 0.0125 0.125 1.25 12500.000125 12.5 [Gossypol equivalents], pg/ml glutamine-gossypol (>125) glutamine-gossypol (>125) tryptophan-gossypol acid-gossypol(>125) amino-benzoic tyrosine-gossypol (>125) tyrosine-gossypol I50 values (the I50 concentrations which caused 50% inhibition ofbinding). 0.8 0.6 0.4 0.2 antibodies). Each point is the average of 2 determinations. The numbers shown in parentheses are Figure 31. ELISA inhibition curves using additional gossypol derivatives for rabbit #15 PAb (polyclonal c/l in in < o © Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [Gossypol equivalents], pg/ml lysine-gossypol (28.31) lysine-gossypol arginine-gossypol (21.51) arginine-gossypol (22.75)ethanolamine-gossypol 2-amino-1-propanol-gossypol (55.05) 3-amino-1-propanol-gossypol (20.86) I50 values (the I50 concentrations which caused 50% inhibition ofbinding). Each point is the average of 2 determinations. The numbers shown in parentheses are Figure 32. ELISA inhibition curves using different gossypol derivatives for MAb (7G2F2G3C6). c/5 c/i IT) SO Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [Gossypol equivalents], pg/ml 0.25 lysine-gossypol (9.93) argine-gossypol (8.41) ethanolamine-gossypol (10.96) 2-amino-1-propanol-gossypol (21.97) 3-amino-1-propanol-gossypol (26.25) I50 values (the I50 concentrations which caused 50% inhibition ofbinding). - - - 0.8 0.6 0.4 - 0.2 Each point is the average of 2 determinations. The numbers shown in parentheses are m co Figure 33. ELISA inhibition curves using different gossypol derivatives for MAb (6G7D11F10F2F8). ■st < o < X <§> X i SJ so Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 250 I 25 “T 2.5 [Gossypol equivalents], pg/ml 0.25 2-amino-1 -propanol-gossypol (18.63) -propanol-gossypol 2-amino-1 (23.99) 3-amino-1-propanol-gossypol argine-gossypol (8.10) argine-gossypol (7.38) ethanolamine-gossypol ■ lysine-gossypol (8.17) lysine-gossypol ■ I50 values (the concentrationsI50 which caused 50% inhibition ofbinding). Each point is the average of 2 determinations. The numbers shown in parentheses are 0 1 0 0.9 0.8 0.7 0.2 0.1 0.6 0.5 0.4 0.3 Figure 34. ELISA inhibition curves using different gossypol derivatives for MAb (6G7D11F10F2H3). in in < iri o < X> Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. potential to differentiate various forms of bound gossypol formed during cottonseed processing. Fresh made gossypol solution (in 10% methanol) was compared with 21 day old gossypol solution in ACC-ELISA using both MAb (6G7D11F10F2F8 and 6G7D11F10F2H3) and PAb (Figure 35 and Figure 36). Freshly made gossypol as standard did not show good competition (Iso>250 fig/ml) comparing with 21 day old gossypol solution (Iso=1.59 (ig/ml) for PAb. Using MAb, however, underivatized gossypol showed no competition when either 21 day old or freshly made gossypol solutions were used at similar concentrations that were used with PAb. During storage, some free aldehydic gossypol may transform into other tautomers such as hemiacetal in methanol (Abdullaev et al., 1990). Gossypol may be oxidized and/or degraded into various products with different properties (Markman, 1968; Nomeir and Abou-Donia, 1982). The gossypol derivatives that formed over the 21 days of aging were apparently reactive to PAb, increasing the effective gossypol concentration for competition. This resulted in a lower Iso value as compared with freshly prepared underivatized gossypol. Storage time seemed to have no effect on competition using MAb, possibly because of their higher specificity. MAb may be able to differentiate Schiff base formed gossypol derivatives and gossypol tautomers, oxidized forms and degraded forms. Isomeric (+)- and (-)-gossypol derivatives were also tested in ACC-ELISA to evaluate the specificity of MAb cell lines (6G7D11F10F2F8 and 6G7D11F10F2H3). Iso values were not significantly different when (+)-gossypol and (-)-gossypol isomers were compared using Tukey’s Studentized Range Test with significance level of 95%. The MAb therefore had a similar affinities or specificities to (+)- and (-)-gossypol 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 0.25 2.5 250 [Gossypol equivalents], pg/ml 0.0250 freshly made freshly gossypolsolution (>250) 21 21 day old gossypol(1.59)solution I50 values (the concentrationsI50 which caused 50% inhibition ofbinding). . - - 0.8 0.6 0.4 - 0.2 antibodies). Each point is the average of 2 determinations. The numbers shown in parentheses are Figure 35. Effects ofgossypol storage time on ELISA inhibition curves for rabbit #15 PAb (polyclonal co < o ■'3’ § X> VO t-ri Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 250 [Gossypol], pg/ml freshly madefreshly gossypolsolution (H3) freshly madefreshly gossypolsolution (F8) 21 21 day old gossypol solution (H3) 21 21 day old gossypolsolution (F8) 0.025 0.25 2.5 0.00250 6G7D11F10F2H3 cell line, respectively. Each point is the average of 2 determinations. 0.8 0.6 0.4 0.2 and 6G7D11F10F2H3). F8 and H3 shown in parentheses represent 6G7D11F10F2F8 cell line and Figure 36. Effects ofgossypol storage time on ELISA inhibition curves for MAb (6G7D11F10F2F8 n c/i t o ■>3- < < XI XI X n v £ > O Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Figure 37 and Figure 38) and did not differentiate between the two isomers. It seems that these MAb recognize the naphthyl group and not the inter-naphthyl bond. A number of statistical approaches have been set up for the definition of detection limit and limit of quantification in immunoassay. However, many users and test kit manufacturers do not use statistical methods, and would like to define the limit detection as the concentration providing either 10% or 15% inhibition of color of development; some researchers define positive samples as providing color development of at least 0.1 OD units less than control (Skerritt, 1995). In this study, the concentration which inhibited color development of 0.1 OD unit comparing with control was defined as the detection limit (Table 4). Table 4. Detection limit for antibodies using gossypol derivatives as competitors Detection limit (pg/ml) Gossypol derivatives Monoclonal antibodies polyclonal antibodies 7G2F2G3 6G7D11F 6G7D11F C6 10F2F8 10F2H3 lysine-gossypol 0.023 8.7 2 .8 1.4 arginine-gossypol 0.03 4.7 2.5 1.4 2 -amino - 1 -propanol-gossypol 0.0025 21.7 4.3 1.7 3 -amino-1 -propanol-gossypol 0.0125 5.5 5.6 4.2 ethanolamine-gossypol 0.05 1 0 .1 3.0 0.9 Usually MAb have better specificity than PAb, so the detection limit is often higher than those based on PAb (Szurdoki et al., 1995). Surprisingly it was found that PAb had much lower I 50 values (Iso values for inhibition curves are summarized in Appendix III) and much lower detection limit than MAb (Table 4) when gossypol 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 250 25 2.5 [Gossypol equivalents], p.g/ml 0.250 (+)-gossypol-2-amino-1 -propanol -propanol (10.67) (+)-gossypol-2-amino-1 (+)-gossypol-lysine (12.72) (+)-gossypol-lysine (13.47) 1-lysine (-)-gossypo (-)-gossypol-2-amino-l-propanol (9.69) in parentheses values are(the concentrationsI50 which caused 50% inhibition ofbinding). for MAb (6G7D11F10F2F8). Each point is the average of4 determinations. The numbers shown - - 6 . 0 0.4 - 0.4 0.2 cA t/i < < 43 O 43 43 Figure 37. Comparison of(+/-)- gossypol derivatives in ACC-ELISA (antibody capture competitive-ELISA) oo VO Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [Gossypol equivalents], pg/ml (-)-gossypol-2-amino-1-propanol (9.09) (+)-gossypol-lysine (10.64) (-)-gossypol-lysine (9.81) (+)-gossypol-2-amino-l-propanol (7.44) in parentheses values are (the concentrationsI50 which caused 50% inhibition ofbinding). 0 0.25 2.5 25 250 for MAb (6G7D11F10F2H3). Each point is the average of4 determinations. The numbers shown 0.4 - t/i t/i in ■sf < < o X X X Figure 38. Comparison of(+/-)- gossypol derivatives in ACC-ELISA (antibody capture competitive-ELISA) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. derivatives with 3-amino-1-propanol, 2-amino-1-propanol, ethanolamine, L-(+)-lysine, and L-(+)-arginine were used as competitors. One reason may be that PAb are less specific, so they cross-react with different forms of gossypol in the reaction mixture (including gossypol derivatives, oxidized gossypol and degraded gossypol formed during reaction). MAb may provide a greater specificity to certain forms of gossypol derivatives in the reaction mixture, therefore requiring higher concentrations of mixed solutions to compete with the solid-phase comparable to PAb. This would result in a lower apparent detection limit when testing a mixture of gossypol derivative products. Ab having broad specificity may be useful in some situations to screen for a family of compounds. Compound-specific Ab may be preferred for other situations where cross reactivity is undesirable. Quite often, high titer PAb give lower detection limits than MAb for compound mixtures because of PAb cross-reactivity (Skerritt, 1995). 3.7: Application of ELISA for the analysis of gossypol in cottonseed products The detection limit was much lower for PAb than MAb, so PAb were adopted first to analyze gossypol in cottonseed samples. L-(+)-lysine was chosen to derivatize gossypol because of the efficiency of the reaction. Seven cottonseed product samples with a variety of Free Gossypol (FG) from 0.08% to 1.12% and Total Gossypol (TG) from 1.06 to 1.35% (Table 5) were selected to be analyzed by ELISA. FG and TG were previously measured (by Dr. M. Calhoun, Texas A&M University) using AOCS official methods (AOCS, Ba 7-58 and Ba 8-78) and (+/-)-gossypol isomers were measured using HPLC. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5: Gossypol contents of cottonseed products* AOCS® HPLC® Sample Treatment description ID* FG% TG% (+)-G (-)-G 2241 Cottonseed meal 0.08 1.06 56.5 43.5 2817 LFFA CS® meats once through expeller 0.33 1.26 57.4 42.6 2822 HFFA CS® meats once through extruder 0.36 1.10 58.2 41.8 2819 HFFA CS meats roasted 20 min 0.91 1.21 58.2 41.8 2820 HFFA CS meats roasted 40 min 0.93 1.26 58.2 41.8 2814 LFFA CS meats roasted 40 min 1.06 1.35 58.2 41.8 2813 LFFA CS meats roasted 20 min 1.12 1.34 57.9 42.1 * samples obtained from M. Calhoun and samp e ID number assignee in the Nutrition/Toxicology Laboratory at the Texas A&M University Agricultural Center, San Angelo, TX; Free Gossypol, Total Gossypol and gossypol isomers measured by M. Calhoun, Texas A &M University; ® low ffee fatty acid cottonseed, ® high free fatty acid cottonseed For TG analysis by ELISA, samples were extracted and derivatized using L-(+)- lysine in 10% methanol. Following 2 h reaction at RT, the extracted slurry was analyzed in ELISA. Results gave poor correlation (r2=0.4332) between the ELISA method and AOCS official method (AOCS, Ba 8-78), and the values were much lower from our ELISA method than from the AOCS official method for the seven samples (Figure 39). One major difference between these two methods is the difference in the extraction and derivatizing reagent used for sample preparation. The AOCS official method (AOCS, Ba 8-78) employs 3-amino-1 -propanol in DMF as complex reagent under 95°C, rather than L-(+)-lysine in 10% methanol at RT in this study. Gossypol is embedded in the cottonseed gland, and the gland consists of an envelope of 2-3 layers of thin-wall tablet shaped cells (Markman, 1968). Even if a sample is ground, it may 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ELISA □ AOCS Sample ID average of4 determinations. 2241 2241 2817 2822 2819 2820 2814 2813 sample delivery) and AOCS official method (Ba 8-78). Each point theis Figure 39. Total Gossypol (TG) in cottonseed samples analyzed by ELISA (10% methanol for TG% t o o Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not break all the glands (wan, 1999). Mild extraction and derivatizing complex reagent, such as L-(+)-lysine in 10% methanol at RT may not efficiently rupture gossypol glands. Moreover, differences in sample processing, hardness, fatty acid content, moisture and purity may influence the grinding efficiency causing the gossypol glands to rupture differently for different samples. Although these all differences in grinding efficiency may also be occurring during sample preparation for the AOCS method, they may not be as apparent because of the strength of the DMF solution. The extracted slurry was directly used in ELISA, and some bound gossypol with protein and other compounds may not be released into this slurry. These all could result in lower values. In order to increase the extraction and derivatization efficiency, samples can be extracted using 3-amino-1-propanol in dimethylformamide (DMF) solution (AOCS, Ba 8-78). However, because of the interference of this solvent in ELISA, the proportion of DMF used in ELISA needs to be investigated and controlled to minimize problems. Our results indicated (Figure 22) that 4% of DMF in non-competitive ELISA could reduce the reactivity of serum by 50% (abs.=l), so the recommended concentration will be lower than this level. The effect of levels of DMF on competitive ELISA should also be investigated. Under ideal conditions, TG measured using ELISA should include all gossypol and gossypol derivatives. Defining what “all gossypol derivatives” includes will prove very challenging. For FG analysis, samples were extracted with 70% aqueous acetone, and filtered. The filtrate was gently purged using nitrogen gas to remove the acetone, then methanol with L-(+)-lysine solution was added. After derivatization, the sample was analyzed in ELISA (Figure 40). FG results obtained by ELISA were lower than FG 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2814 2813 2820 Sample ID point is the average of4 determinations. 2817 2822 2819 □ AOCS (x) ■ ELISA (y) Y=0.5569x-0.0045 r^O.69 2241 methanol for sample delivery) and AOCS official method (Ba 7-58). Each - - - 8 6 Figure 40. Free Gossypol (FG) in cottonseed samples measured using ELISA (10% 2 . . . 0 0 0.4- 0 FG% o •fc. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. results from AOCS method (Ba 7-58). After removing the acetone, the solutions became cloudy. Water is a poor solvent for gossypol, therefore some gossypol may have crystallized from the solution. After adding L-(+)-lysine, gossypol may not have been completely derivatized. Underivatized gossypol could decrease the effective gossypol concentration in ACC-ELISA, resulting in lower gossypol values as compared to the results obtained using the AOCS official method. Because of the poor solubility of gossypol in water, the above method for FG analysis was modified. FG was extracted using 70% acetone and filtered (AOCS, Ba 7- 58), and L-(+)-lysine in PBS was slowly added into filtrate to dilute the acetone concentration from 70% to 10%. Following 2 h reaction at RT, the samples were analyzed using ELISA. The results showed good correlation (^=0.96) between our ELISA and AOCS official method (Figure 41, Appendix IV) under the conditions of the method. However the results from ELISA were approximately two times higher than those determined using AOCS methods. It is possible that some gossypol was oxidized and/or degraded during sample storage and shipment. Loss of the aldehyde groups would result in nondetection by the AOCS official method (AOCS, Ba 7-58). The ELISA, however, may still detect these products, depending on their reactivity to the Ab binding sites and their extractabilities. Free Gossypol measured by ELISA method could therefore be defined as gossypol and gossypol derivatives which are soluble in 70% acetone and have affinity for the PAb used in the assay. Bound Gossypol (BG) can be determined mathematically, and BG can be defined as gossypol derivatives which could not be extracted by 70% acetone but have specific binding sites for PAb used in the assay. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 2813 2814 Sample ID 2822 2819 2820 2817 y=2.0885x rMJ.96 determinations except sample 2241 which is the average of 5 determinations. □ AOCS (x) ■ ELISA (y) sample delivery) and AOCS official method (Ba 7-58). Each point is the average of 2241 Figure 41. Free Gossypol (FG) in cottonseed samples determined using ELISA (10% acetone for 1.5 1.5 - 2.5 - 0.5 - FG% n o O Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.8: Future research In this study, anti-gossypol PAb and MAb were successfully produced, and the model system studies showed that PAb and MAb could recognize gossypol derivatives formed via Schiff bases. Preliminary research on gossypol analyses of cottonseed products appeared to be a promising and challenging research. In order to produce and characterize different MAb cell lines with different specificities, different forms of relatively pure gossypol and gossypol derivatives (including gossypol derivatives formed via a Schiff base intermediate and via phenol hydroxy group condensation, oxidized gossypol, degraded gossypol, (+)- and (-)- gossypol, and (+)- and (-)-gossypol derivatives) need to be made to produce, screen and select hybridomas, and to evaluate the affinities/specificities of the Ab produced. MAb with different specificities should be adapted and optimized in an immunoassay to rapidly and accurately measure the quantity of gossypol. This could potentially be adapted to a kit format for use by the cottonseed industry, veterinarians and plant breeders. The immunoassay results need to be correlated with established AOCS colorimetric methods, HPLC, and bioavailability measurements using a large number and variety of samples. The advantages of specific binding between Ab-Ag for measuring different forms of gossypol, and should be further exploited to improve our knowledge of the forms and bioactivity of bound gossypol. MAb having specific affinity to either (+)- or (-)-gossypol could provide an alternative method to isolate gossypol isomers. The available methods so far reported to separate gossypol isomers include HPLC method after conversion of gossypol into diastereomeric Schiff-bases (Matlin, et al., 1987) and crystallization from acetone 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Dowd, 1998). (+)-Gossypol and (-)-gossypol have different biological activities, and the more potent anti-spermatogenic, anti-tumor and anti-HIV properties of (-)-gossypol, have led to a requirement for large quantities of this specific isomer for further biological studies. Immunoaffinity columns using MAb have been developed commercially in the United States, the United Kingdom (Scott and Trucksess, 1997) and Canada for the isolation of many types of analytes. The current research demonstrated the production of antibodies to gossypol. The potential uses of this technology are many, from quality control test kits by use in industry, to research tools to better understand the physiological effects of gossypol. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4 SUMMARY AND CONCLUSIONS The development of immunochemical methods provided a potential tool to study gossypol chemistry in model systems and to analyze gossypol in cottonseed products. Protein-gossypol conjugates and gossypol derivatives were produced via Schiff bases, and the addition of stabilizing reagent NaBHsCN favored Schiff base formation and increased product yield. High purity (+)- and (-)-gossypol isomers (> 99%) were obtained through gossypol crystallization from acetone. Polyclonal antibodies (PAb) against gossypol could be produced when rabbits were immunized with LPH-gossypol immunogens. The preparation methods for immunogens were found to greatly affect antibody production. Assay parameters, such as microtiter plates, incubation time and temperature for Ab-Ag interactions, dilution buffers for 1 st and 2 nd antibodies, and blocking buffers could greatly influence the ELISA. Monoclonal antibodies (MAb) were developed by fusion of immune mouse splenocytes and NS-1 myelomas under PEG-1500 fusion buffer. PAb and MAb were used in an antibody capture competitive-ELISA and different forms of gossypol were used to evaluate the antibodies for their sensitivities and specificities. Under optimum conditions, approximately 0.2 (ig/ml of gossypol equivalents caused 50% binding inhibition (I 50) using rabbit PAb, and 7 to 55 (ig/ml of gossypol equivalents caused 50% binding inhibition of MAb to the solid-phase (BSA- G), when gossypol derivatives with L-(+)-lysine, L-(+)-arginine, L-2-amino-l-propanol, 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3-amino-1-propanol and ethanolamine were used competitively. The MAb cell lines obtained had equal affinity/specificity for (+)- and (-)-gossypol isomeric derivatives. Underivatized gossypol as competitor gave much higher I 50 values for PAbs and showed no competition for MAb, indicating that either the antibodies showed low or no recognition of unbound gossypol or that gossypol reacted non-specifically to reagent proteins. Gossypol samples were analyzed using PAb in an antibody capture competitive- ELISA. Preliminary research indicated that extraction and derivatization methods for sample delivery could greatly influence the results. After improvement of the method, Freel Gossypol analysis gave good correlation (^=0.96) between ELISA and AOCS official methods, so ELISA methods seem to have potential to provide a rapid and accurate method for gossypol analysis. 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES Abdullaev, N.D., Tyshchenko, A.A., Nazarova, L.P. 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APPENDIX I Competition after 1st cloning using different gossypol derivatives (250 pg/ml of gossypol equivalent) as competitors Cell line Gossypol-derivatives A/Ao Lysine-g 0.181/0.958 Arginine-g 0.198/0.958 6G7D11 Amino-propanol-g 0.161/0.958 Ethanolamine-g 0.151/0.958 Lysine-g 0.157/0.488 Arginine-g 0.141/0.488 6G7D2 Amino-propanol-g 0.150/0.488 Ethanolamine-g 0.153/0.488 Lysine-g 0.158/0.400 Arginine-g 0.177/0.400 6G7D7 Amino-propanol-g 0.153/0.400 Ethanolamine-g 0.156/0.400 Lysine-g 0.161/0.393 6G7G3 Arginine-g 0.193/0.393 Amino-propanol-g 0.148/0.393 Ethanolamine-g 0.152/0.393 Lysine-g 0.162/0.404 6G7B6 Arginine-g 0.185/0.404 Amino-propanol-g 0.167/0.404 Ethanolamine-g 0.169/0.404 Lysine-g 0.142/0.325 6G7D12 Arginine-g 0.175/0.325 Amino-propanol-g 0.149/0.325 Ethanolamine-g 0.168/0.325 (table cont.) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lysine-g 0.268/2.506 5D7D3 Arginine-g 0.333/2.506 Amino-propanol-g 0.198/2.506 Ethanolamine-g 0.186/2.506 Lysine-g 0.285/2.775 5D7E3 Arginine-g 0.256/2.775 Amino-propanol-g 0.199/2.775 Ethanolamine-g 0.186/2.775 Lysine-g 0.170/0.778 5D7F3 Arginine-g 0.155/0.778 Amino-propanol-g 0.154/0.778 Ethanolamine-g 0.149/0.778 Lysine-g 0.252/1.951 5D7E4 Arginine-g 0.223/1.951 Amino-propano 1-g 0.205/1.951 Ethanolamine-g 0.192/1.951 Lysine-g 0.274/1.964 5D7F4 Arginine-g 0.273/1.964 Amino-propanol-g 0.182/1.964 Ethanolamine-g 0.218/1.964 Lysine-g 0.282/1.736 5D7G4 Arginine-g 0.271/1.736 Amino-propanol-g 0.207/1.736 Ethanolamine-g 0.228/1.736 (table cont.) 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lysine-g 0.182/0.239 7G2A5 Arginine-g 0.173/0.239 Amino-propanol-g 0.160/0.239 Ethanolamine-g 0.171/0.239 Lysine-g 0.178/0.280 7G2B5 Arginine-g 0.178/0.280 Amino-propanol-g 0.175/0.280 Ethanolamine-g 0.178/0.280 Lysine-g 0.189/0.407 7G2A6 Arginine-g 0.181/0.407 Amino-propanol-g 0.179/0.407 Ethanolamine-g 0.184/0.407 7G2F2 Arginine-g 0.131/0.644 5H9F8 Arginine-g 0.136/0.344 Note: g represents gossypol. 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX II Peak results of HPLC (high performance liquid chromatography) for (+)- and (-)- gossypol isomers Name Area Purity (%) (+)-gossypol: 80089 (-)- 99.63 gossypol (-)-gossypol: 22017381 (+)-gossypol: 9784542 (+)- 99.96 gossypol (-)-gossypol: 3802 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX III I5 0 values for gossypol and gossypol derivatives I 5 0 Values (pg/ml) Gossypol derivatives MAb PAb 7G2F2G3C 6G7D11F10 6G7D11F 6 F2F8 10F2H3 Lysine-gossypol 0.197 28.31 9.93 8.17 Arginine-gossypol 0.209 21.51 8.41 8.10 2-amino-1 -propanol-gossypol 0.195 55.05 21.97 18.63 3-amino-1 -propanol-gossypol 0.174 20.86 26.25 23.99 Ethanolamine-gossypol 0.225 22.75 10.96 7.38 fresh made gossypol >250 >250 >250 >250 21 day old gossypol 1.59 >250 >250 >250 Glutamine-gossypol >125 - -- T yrosine-gossypol >125 - -- T ryptophane-gossypol >125 - -- Para-amino-benzoic acid- >125 - - - gossypol (+)-gossypol-lysine - - 12.72 10.64 (-)-gossypol-lysine - - 13.47 9.81 (+)-gossypol-2-amino-1 - - - 9.08 7.44 propanol (-)-gossypol-2-amino-1 - - - 9.53 9.09 propanol 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX IV Comparison of AOCS and ELISA methods for FG analysis Sample ID* FG% (AOCS:x) FG% (ELISAry) 2241 0.08 0.35 2817 0.33 0.69 2822 0.36 0.9 2819 0.91 2.0 2820 0.93 1.95 2814 1.06 1.94 2813 1.12 2.45 * samples obtained from M. Calhoun (Texas A&M University) and sample ID number assigned in the Nutrition/Toxicology Laboratory at the Texas A&M University Agricultural Center, San Angelo, TX. FG (x) measured by M. Calhoun (Texas A&M University) and FG (y) measured by ELISA method. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA The author was bom in Shandong Province, People’s Republic of China, on August 27, 1968. She graduated from the Department of Cereal and Oil Science and Engineering, Wuxi Institute of Light Industry, with a bachelor’s degree, in 1991. As a top ranking student, she was recommended for further study as a graduate student in the same department under Professor Hongsun Diao. Her study area included oilseed processing, oil modification, unit design, and oilseed by-product utilization (including vitamin E, tannin, phytic acid, oryzanol and gossypol). In 1995, she was admitted as a doctoral candidate under Dr. Leslie C. Plhak in the Department of Food Science, Louisiana State University, with the research on compound analysis using immunochemical methods. Currently, she is studying in Food Science for the degree of Doctor of Philosophy, which will be awarded in August 1999. 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DOCTORAL EXAMINATION AND DISSERTATION REPORT Candidate: Xi Wang Major Pield: Food Science Title of Dissertation: Development of Immunochemical Methods for Gossypol Analysis Approved: Mai Professor ( ^ / Dead 'of the"Graduate School EXAMINING COMMITTEE: Date of Examination: April 29, 1999 Reproduced with permission of the copyright owner. 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