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Developmental of a novel affinity ELISA and ita application PDF

171 Pages·2012·7.54 MB·English
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Preview Developmental of a novel affinity ELISA and ita application

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

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reports examining affinity maturation in rainbow trout failed to show . PBL cultures. 106. 25. Representative three dimensional histogram of antibody affinities from TNP-LPS and LPS stimulated anterior kidney cultures. 106 dosed with 50 ul cold RPMI-1640, 10 Ill diluted steelhead serum in 1xMBB.
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