Developmental of a novel affinity ELISA and ita application

171 Pages·2012·7.54 MB·English

by  Shapiro, David A.

AN ABSTRACT OF THE THESIS OF

David A. Shapiro for the degree of Doctor of Philosophy in Microbiology presented on
December 7, 1995. Title: Development of a Novel Affinity ELISA and Its Application to
the Analysis of Affinity Maturation in Trout.
Redacted for Privacy
Abstract approved:
Stephen L. Kaattari
This study was undertaken to develop a solid phase, enzyme based
immunoassay to characterize affinity maturation and antibody affinity
heterogeneity in rainbow trout (Oncorhynchus mykiss). The few existing
reports examining affinity maturation in rainbow trout failed to show
changes in antibody affinity during an immune response. A competitive
ELISA was established in which the binding of serum or (leukocyte) culture
supernatant antibodies to serially decreasing, plate-bound, multi-haptenated
antigen is inhibited by graded doses of a free hapten inhibitor. The assay
yields an estimate of the distribution of affinities within polyclonal mixtures
of antibodies, as well as estimates of the apparent affinity constants (aK) for
each antibody subpopulation. In addition, a method for the calculation of an
average affinity (K) from affinity subpopulation data is described. The assay
was used to characterize increases in average affinity during the primary and
secondary immune response to both a T-dependent form of the
trinitrophenol hapten (TNP-KLH) and a T-independent form (TNP-LPS) in
trout. Significant increases in affinity were documented as early as three
weeks post immunization in these animals. The sensitivity of the assay
allowed detection of small differences in average antibody affinity produced
against a T-dependent antigen in populations of rainbow trout embryonically
exposed to 1 ppm aflatoxin B1 versus controls. The ELISA was also adapted
for use in measuring leukocyte culture antibody affinities. Armed with this
tool, it became possible to demonstrate antigen specific stimulation of high
affinity B cell clones in vitro, suggesting that the mechanism of affinity
maturation in vivo in rainbow trout may be similar to that previously
described in mammals. Finally, murine IgM and IgG anti-TNP antibodies
were characterized during the first three weeks of an immune response to
TNP-KLH.
Development of a Novel Affinity ELISA and Its Application to the
Analysis of Affinity Maturation in Trout
by
David A. Shapiro
A THESIS

submitted to

Oregon State University

in partial fulfillment of

the requirements for the

degree of

Doctor of Philosophy

Completed December 7, 1995

Commencement June 1996
Doctor of Philosophy thesis of David A. Shapiro presented on December 7, 1995.
APPROVED:
Redacted for Privacy
Major Professor, representing Microbiology
Redacted for Privacy
Chaiiman of the Department of Microbiology
Redacted for Privacy
Dean of Graduate School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for Privacy
David A. Shapiro, Author
ACKNOWLEDGMENTS
I gratefully acknowledge the support of my thesis advisor, Dr. Stephen L. Kaattari
for his years of mentorship at Oregon State University. I would also like to thank the
Department of Microbiology, Oregon State University for their patience and assistance with
all matters pertaining to my research during my residence in Virginia. Finally, I would like
to thank Dr. M. Adkison and Dr. C. Ottinger for their advice and assistance during the
development of the ELISA assays and during data collection. Further assistance with data
collection was provided by L. Gilkey, J. Bishop, W. Morgan, J. Klemer, M. Bourbano,
and H. Christensen. This work was supported by N.I.E.H.S. grant #ES057383.
TABLE OF CONTENTS

Page
INTRODUCTION
1

LITERATURE REVIEW 3

The Generation of Antibody Binding Site Diversity 3

Avidity vs Affinity 5

Factors Influencing Affinity Maturation
6

Rainbow Trout Immunoglobulins and the Genes Encoding Them 10

Antibody Affinity at the Molecular Level 12

The Relationship Between Free Energy and Equilibrium Constants 17

Empirical Determination of Affinity Constants 18

Equilibrium Dialysis 19

Fluorescence Quenching 25

Inhibition of the Plaque Forming Cell Response 27

Precipitation Techniques 30

The Farr Technique 30

Hapten Inhibition of Precipitation 33

Solid Phase Assays 34

MATERIALS AND METHODS 38

Animals 38

Antigen Preparation 39

Immunizations 41

Plasma, Sera, and Leukocyte Harvesting 42

Culture of Trout Leukocytes 43

Measurement of the anti-TNP Antibody Forming Cell (AFC)

Response 45

Titration of Anti-TNP Activity and Total Immunoglobulin in

Serum and Plasma by ELISA. 47

Monoclonal Antibody Production. 49

Screening of Monoclonal Antibodies 51

ELISA 51

Polyacrylamide Gel Electrophoresis and Immunoblotting 51

SDS-PAGE 51

2D-IEF 53

Immunopurification of Antibodies (Trout and Murine) 53

Gel Fractionation of Murine IgG and IgM 55

Detection of murine IgM and IgG 56

Determination of Plate Antigen Saturation 57
TABLE OF CONTENTS (Continued)
Page
Determination of Antibody Concentration for Affinity Analysis 58

The Affinity ELISA 59

Determination of Antibody Subpopulation Sizes 62

Determination of Antibody Subpopulation Affinities 63

Assay Repeatability 64

Analysis of Antibody From Trout Lymphocyte Culture 65

Analysis of Murine Serum Antibody Affinities 65

Aflatoxin Experiments 66

RESULTS 67

Development of the ELISA-Based Affinity Assay 67

Introduction 67

Recognition of All H Chains by 1.14 67

Development of Alternate Anti-Trout Monoclonal

Antibodies 68

Plate Antigen Saturation 71

Typical Affinity Profiles 75

Determination of Plate Coating Antigen Concentration 75

Assay Repeatability 78

Alternate Ways of Expressing Antibody Affinity Data 79

Assessment of Affinity Maturation in Rainbow Trout 83

Affinity Distributions During Primary and Secondary

Responses to TNP-KLH 83

Early Resolution of Affinity Maturation (3 week) 85

Application of the Affinity Assay to a Pathologic Situation:

AFB1 Immunotoxicity 88

The Effect of Immunogen Concentration on Antibody Affinity 94

Introduction 94

The Effects of Exogenous TNP-LPS on Anti-TNP Titer 94

In Vitro Dose Response Curves 95

Dose Response by ELISA 95

Dose response by the enumeration of plaque

forming cells (PFC) 98
TABLE OF CONTENTS (Continued)
Page
Dose Dependent Production of High Affinity Antibodies In

Vitro 100

In Vitro Antibody Affinity Profiles 100

Comparison of Mammalian Multimeric and Monomeric
Antibodies With Trout Ig 109

Separation of Murine IgM and IgG Immunoglobulins 109

Comparison of Murine IgM and IgG Affinity Profiles 111

DISCUSSION 119

SUMMARY AND CONCLUSION 129

BIBLIOGRAPHY 131

APPENDICES 143

APPENDIX I: BUFFERS AND REAGENTS 144

APPENDIX II: SAMPLE AFFINITY ELISA CALCULATIONS 150
LIST OF FIGURES

Figure Page
Scatchard analysis of the binding of antigen to
1
monoclonal and polyclonal antibodies 23
2 Sips plot of a purified anti-TNP antibody preparation 28
3 Anti-DNP-BGG affinity distribution in C57L/J mice
by age 31
4 Sample microtiter plate layout 61
5 Sample inhibition curves 63
Two-dimensional electrophoretic separation of
6
anti-TNP antibodies from a single rainbow trout
immunized with TNP-KLH 69
7 Two-dimensional electrophoretic separation of
anti-TNP antibodies from a mixture of trout antisera
immunized with TNP-KLH 70
Staining of rainbow trout antibodies by two
8
representative monoclonal antibodies, 12F8 and
9H12 72
9 Determination of surface saturating antigen
concentration by peroxidase saturation 74
Typical antibody affinity distribution from a rainbow
10
trout anti TNP serum 76
11 Effects of improper plate antigen coating
concentrations 77
ELISA affinity analyses (A, B, C) of a trout anti-TNP
12
serum 81
Histograms depicting the percentage of total antibody
13
within arbitrary aK ranges 82
LIST OF FIGURES (Continued)
Figure Page
14 Three dimensional histogram analysis 84
Affinity maturation of the rainbow trout anti-TNP
15
response 86
16 Affinity distributions at three weeks post
immunization in trout immunized with TNP-KLH in
Freund's Complete Adjuvant. 89
17 The effect of exogenous TNP-LPS on measurement
of anti-TNP titer in leukocyte culture supernatants 96
18 PBL and AK dose response curves 97
19 In vitro TNP-LPS plaque forming cell dose response 99
20 Effect of TNP-LPS dose on peripheral blood leukocyte
in vitro antibody affinity generation 101
21 Effect of TNP-LPS dose on anterior kidney in vitro
antibody affinity generation 101
22 Effect of LPS dose on peripheral blood leukocyte
in vitro antibody affinity generation 101
23 Effect of LPS dose on anterior kidney in vitro
antibody affinity generation 101
Representative three dimensional histogram of
24
antibody affinities from TNP-LPS and LPS stimulated
PBL cultures 106
Representative three dimensional histogram of
25
antibody affinities from TNP-LPS and LPS stimulated
anterior kidney cultures 106