The relationship between antibody redox structure and affinity in rainbow trout

243 Pages·2017·5.27 MB·English

by  Jianmin Ye

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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects
2008
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Jianmin Ye
College of William and Mary - Virginia Institute of Marine Science
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Ye, Jianmin, "The relationship between antibody redox structure and affinity in rainbow trout" (2008).
Dissertations, Theses, and Masters Projects. Paper 1539616918.
https://dx.doi.org/doi:10.25773/v5-ddjh-pz29
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The Relationship between Antibody Redox Structure
and Affinity in Rainbow Trout
A Dissertation
Presented to
The Faculty of the School of Marine Science
The College of William and Mary in Virginia
In Partial Fulfillment
Of the Requirements for the Degree of
Doctor of Philosophy
by
Jianmin Ye
2008
APPROVAL SHEET
This dissertation is submitted in partial fulfillment of
The requirements for the degree of
Doctor of Philosophy
Jianmin Ye
Approved, by the Committee, August, 2008
~i(}~2)
CStemph1enC Lh. ~atitnanria,n P/h .D.
Advisor
l~--7
\~LL.-.~
Hany W. Dickerson, Ph.D.
University of Georgia
Athens, GA
11
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ..................................................................... vii
LIST OF TABLES ................................................................................v iii
LIST OF FIGURES ................................................................................ .ix
ABSTRACT ........................................................................................x ii
INTRODUCTION ...................................................................................2
Antibody Structure and Function ............................................................... .2
General Structure of an Ig Monomer (IgG) ...............................................2
Basic Teleost Antibody Structure ........................................................... 6
Structural Classes of Antibodies (I so types) ............................................. .11
Evidence for Teleost I so types ............................................................. 19
Redox Structure of Teleost and Mammalian Antibodies .............................. 23
Molecular Basis for Binding Site Diversity ............................................. 32
Antibody Affinity ........................................................................... 36
Antibody Avidity ........................................................................... 41
Teleost Antibody Affinity and Avidity .................................................. .43
Affinity Maturation .........................................................................4 4
Teleost Antibody Affinity Maturation .................................................. .46
Antibody Secreting Cell (B Cell) ........................................................ .48
B cell Activation ....................................................................... 50
Location ofA ntigen Stimulation ..................................................... 52
Antibody Synthesis .................................................................... 53
Melding Past and Current Redox Studies ................................................... 55
MATERIALS AND METHODS ................................................................. 62
Animals ........................................................................................... 62
Antigen Preparation ............................................................................ 62
111
Immunization (Trout) .......................................................................... 64
Serum and Plasma Preparation ............................................................... 64
Preparation of Intracellular Immunoglobulin ............................................... 65
Biotinylation of Antibodies I Immunoglobulins ............................................ 65
Antibody and Immunoglobulin Quantification (qELISA) ................................. 66
Affinity Partitioning Analysis (Affinity ELISA) ...........................................6 7
Gel Electrophoresis and lmmunoblotting ................................................... 69
Purification of Trout Antibodies I Immunoglobulin ....................................... 70
Immunoadsorbent Preparation and Antibody Binding Capacities ................... 70
Immunoadsorption ofanti-TNP Antibodies ............................................ 70
Immunoadsorption ofA nti-TNP Antibodies Using Carriers with Varying
Haptenation Ratios .......................................................................7 1
Isolation ofI mmunoglobulin by S-300 Size Exclusion Chromatograph .......... 72
Disulfide Bond (DSB) Coefficient Determination ......................................... 73
Dithiobis (Succinimidyl) Propionate (DSP) Antibody Analysis ......................... 73
Trout Antibody I Ig Half-life Analysis ...................................................... 74
In Vitro Immunization of Trout Lymphocytes .............................................. 75
Antigen Pulsing Protocol ...................................................................... 76
Mouse Immunizations and Monoclonal Antibody (mAb) Production .................. 7 6
RESULTS ........................................................................................... 78
Section I The Association between Affinity and Redox Structure within Individual
Antisera ................................................................................. 78
1.1. The Use of Selective Immunoadsorption to Determine the Relationship
between Antibody Redox Structure and Affinity ........................ 78
Section 2 Relationship between Inter-monomeric Disulfide Polymerization and
Affinity Maturation ................................................................... 84
2.1. Association of Antibody Redox Structure and Affinity in the
Process of Affinity Maturation ............................................. 84
Section 3 In Vitro Induction of High Affinity Antibodies and the Impact on Redox
Structure (The need for antigen pulsing when analyzing in vitro induced
antibodies) .............................................................................. 89
IV
3.1. Stimulatory Concentrations ofTNP-LPS Inhibit Supernatant Anti-TNP
Antibodies ......................................................................9 4
3.2. Estimation ofTNP-LPS in Supernatant by an Inhibition ELISA ........ 101
3.3. Association of Affinity with the Degree ofDisulfide Polymerization
among In Vitro Induced Antibodies ...................................... 10 1
3.4. Comparison of Antibody Titer and Affinity of Pulsed and Control
Groups ........................................................................ 108
Section 4 Effect of Redox Structure on Antibody Half-life ............................ .108
4.1. Assessment ofPurified Anti-TNP Stability In Vitro ...................... 113
4.2. In Vivo Assessement of Anti-TNP Half-life (Modulation of Affinity and
Redox Forms) ................................................................. 113
4.3. In Vivo Assessment of Non-immune, Whole Ig Half-life (Modulation of
Redox Forms) ................................................................. 116
4.3.1. Effect ofBiotinylation on Immunoglobulin Half-life .............. 123
4.3.2. Half-life ofBiotinylated Whole Immunoglobulin In Vivo ......... 123
Section 5 Effect of Polymerization on Antibody Affinity (Trout and Murine
Studies) ................................................................................ 130
5.1. Comparison of Intracellular vs. Secreted Antibody Affinity and
Structure ....................................................................... 130
5.2. Effect of Reduction via 2-Mercaptoethano1 on Antibody Affinity and
Structure ....................................................................... 13 8
5.2.1. In Vitro Culture of Murine Hybridomas under Reductive
Conditions ................................................................ 138
5.2.2. In Vitro Induction of Trout Lymphocytes under Reductive
Conditions ................................................................ 143
DISCUSSION ..................................................................................... 154
Relationship between Antibody Affinity and the Degree of Disulfide
Polymerization ................................................................................ 155
Determination o.fA ntibody Redox Structure and Affinity within Antisera by
Selective Immunoadsorption ............................................................. 155
Relationship between Affinity Maturation and Redox Structure ................... 157
v
Affinity Driven Selection and Redox Structure ....................................... 160
The Individuality of the Redox I Affinity Relationship ................................... 164
Affinity-Determined Redox Forms Affect Antibody Half-li£e .......................... 165
The Effect of Polymerization on Antibody Affinity ...................................... 168
Comparison ofI ntracellular vs. Secreted Antibody Affinity and Structure ....... 170
Effect ofI n Vitro Reduction on Antibody Affinity and Structure .................. 172
SUMMARY and FUTURE STUDIES ......................................................... 174
LITERATURE CITED ............................................................................ 176
APPENDICES ..................................................................................... 201
Appendix A Solutions and Reagents .......................................................... .20 1
Appendix B Affinity Partitioning Assay (Affinity ELISA) .................................2 05
Appendix C SDS-CAGE Recipes .............................................................. 211
Appendix D TNP-lysine Conjugated CNBr Activated Sepharose Beads .................2 12
Appendix E Determination of the Binding Capacities ofDifferent Density TNP-BSA
Conjugated Beads .................................................................................2 14
Appendix F Supplementary Data Table 1-3 .................................................. .219
Appendix G Kinetics of Affinity Maturation ....................................................... 222
Appendix H Development of an Inhibition ELISA to Estimate 1NP-LPS in Sup .......2 27
VITA ................................................................................................2 30
Vl
ACKNOWLEDGMENTS
So many people, in these six years that I spent at VIMS pursuing my Ph.D degree, have
made the completion of my final dissertation possible. I would like to acknowledge and
express my great gratitude from the bottom of my heart to these people.
First of the first, I wish to thank my advisor (major professor), Dr. Stephen L. Kaattari.
He helped me find a great topic and collect needed information, brought me inspiration
and led me toward many exciting fields, and kept encouraging me to "keep fighting"
when I had hard time on my research. He is in my memory always with me. He made
sure that I was on the right track toward my scientific goals, and the completion of my
final thesis. His latest vocation was to take time to edit my papers and polish my English.
All these things have meant a lot to me and I will keep my appreciation for him in my
heart forever.
Secondly, I wish to thank all the people in the Kaattari immunology laboratory. I enjoyed
working with each ofthem on many exciting projects. We learned from each other and
cooperated to complete several exciting projects, and we are still doing so now. Such a
friendly and healthy scientific environment provided me with a friendly, family
atmosphere but also a healthy, stimulating, research environment. All ofthis greatly
contributed to an enjoyable six years of student life here. I appreciate all oftheir
friendliness and spirit of cooperation and assistance.
I am grateful to my committee members (Drs. Bromage, Zwollo, Dickerson and Reece)
for their thoughtful comments and suggestions. Particularly, I want to thank Dr. Erin S.
Bromage for his timely help and his sharp insight during our interactions on our projects.
Dr. Bromage was a post-doctoral associate in Dr. Kaattari's lab when I came, now he is a
professor at University of Massachusetts Dartmouth. He showed me the basic ABC's of
research till I demonstrated a great degree of independence. From then on, we worked
closely on our projects. Our interactions were always exciting and we generated great
ideas which extensively contributed to my research. For this, I will always be thankful.
Most especially, to my wife (Ying Zhang) and my parents (Dintao Y e and Aihua Huang).
They are always with me no matter where they are. Their trust in me made me stronger
and confident to face challenges and complete my dissertation.
Thank again for all those who helped me. Without them, I could not have completed my
dissertation.
Vll
LIST OF TABLES
Table Page
1. Densitometric analyses of the redox structure of affinity-fractionated antibodies
from three additional individual sera ................................................. .219
2. Titer, affinity and redox structure during the affinity maturation ofthe antibody
response to TNP-KLH from the five individual fish ................................ .220
3. Titer, affinity and redox structure of in vitro induced antibodies .................. 221
4. Optimization of the TNP-LPS detection assay .......................................2 28
5. A theoretical projection of the effect of washing on regulation ofTNP-LPS
concentration ..............................................................................2 29
vm
LIST OF FIGURES
Figure Page
I. Schematic of the classical monomeric JgG molecule ................................. 3
2. Schematic of teleost IgM .................................................................. 7
3. Comparison ofthe V and C region domains of the teleost heavy chain compared
with similar domains from other vertebrate H chains ................................. 9
4. Schematic ofthe mammalian, elasmobranch and teleost Jg classes of secretory
and transmembrane forms ................................................................ 13
5. Regions of mammalian Fcs assoicated with specific effector functions .......... .17
6. IgH loci of zebrafish and rainbow trout ................................................ 21
7. Schematic of rainbow trout IgM redox structure ..................................... .24
8. Electrophoretic behavior of human, rat, and teleost Igs ............................. .27
9. Disulfide linkages in the CH4 and C-terminal region of catfish J-1 chain ........... .29
I 0. IgH gene rearrangement and somatic mutation in mammals ........................ 34
11. Scatchard plot for antibody affinity analysis .......................................... 39
12. Antibody dono types share structural diversity ......................................... 56
13. Structure of supernatant antibodies and lysate Igs in vitro ........................... 60
14. Affinity fractionation of trout serum antibodies ........................................ 80
15. Redox structural profiles of affinity-purified antibodies ............................. 82
16. Relationship between affinity and disulfide binding among individual trout ..... 85
17. Antibody titers and affinities induced to TNP-KLH ................................. 87
18. Relationship between average affinities and DSB coefficients during affinity
maturation ................................................................................... 90
19. Relationship between disulfide polymerization and affinity maturation ........... 92
20. Antibody titers and average affinities of non-pulsed (control) cultures ............ 95
21. Effect of exogenous antigen (TNP-LPS) on anti-TNP detection .................... 97
22. Effect of exogenous antigen (TNP-LPS) on anti-TNP affinity analysis ............ 99
23. Estimation ofTNP-LPS concentrations in pulsed and washed culture
supernatant ............................................................................... I 02
ix