IgG subclasses, specific antibodies and immunoglobulin allotypes in children with invasive

503 Pages·2017·13.2 MB·English

IgG Subclasses, Specific Antibodies and Immunoglobulin
Allotypes in Children with Invasive Haemophilus Influenzae
type B and Staphylococcus Aureus Infections
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Thesis Presented for the Degree of o
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DOCTOR OF PHILOSOPHY
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in the Department of Paedi atrics and Child Health
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UNIVERtSITY OF CAPE TOWN
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by
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DR ELIZABETH ANNE GODDARD
March 1994
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The copyright of this thesis vests in the author. No
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quotation from it or information derived from it is to be

published without full acknowledgeement of the source.
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The thesis is to be used for private study or non-
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commercial research purposes only.

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Published by the Universit y of Cape Town (UCT) in terms
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of the non-exclusive license granted to UCT by the author.
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IgG SUBCLASSES, SPECIFIC
ANTIBODIES AND
IMMUNOGLOBULIN ALLOTYPES
IN CHILDREN WITH INVASIVE
HAEMOPHILUS INFLUENZAE TYPE B
AND
STAPHYLOCOCCUSAUREUS
INFECTIONS
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ACKNOWLEDGEMENTS
This thesis has finally come together through the help, support and encouragement of many
friends. I acknowledge the substantial contribution made by a number of people m
enabling me to complete this thesis and thank them all. In particular special thanks to:
My supervisor, Professor David Beatty for giving me this opportunity, initiating my
interest in immunology and allowing me the freedom to pursue academic interests and
avenues. I also thank him for his guidance, encouragement and constructive criticism.
The members of Professor Beatty's Immunology Service: Jane Hughes, Margaret Cooper,
Erica Malan, Sandy Pienaar, Kathy Wood, Renee Allin and Irene Mardarowicz. I thank
them all for their friendship, kindness, moral support and for all their help in tirelessly
loading ELISA plates.
Mrs Lynore Heuer, who found the time to type this thesis, decoding and typing many
pages of illegible scrawl and transforming it into a manuscript.
Dr Carolyn Kewley for her friendship and for the hours she spent proof reading this thesis.
Dr Derek Chalton of the South African Medical Research Council, Division of Biostatistics
for his help with the Box Cox transformation of the IgG subclass data.
Professor Michael Mann for patiently guiding me through the basics of statistics. Dr
Marinda Smit for helping me plug in data and whose mastery of the statgraphics
programme was invaluable.
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Professor MW Turner, Molecular Immunology Unit, Institute of Child Health, University
of London, for measuring mannose binding protein on selected specimens on selected
specimens.
Dr P Creemers, Department of Tissue Immunology, University of Cape Town for
measuring C4 isotypes on selected specimens.
Dr Ed Merrifield for his help with the HPLC analysis of the Haemophilus influenzae type b
polyribose phosphate.
Dr E B Hoffman for collection of the serum samples from patients with
osteomyelitis/septic arthritis.
To the children and their parents who participated in this study.
To my parents who have always supported me. To Don, for his constant encouragement,
never ending support and great patience over the years.
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TABLE OF CONTENTS
Page
Title page
Acknowledgements i
Contents 111
List of Tables xii
List of Figures XVl
Abbreviations XXVl
Abstract XXlX
SECTION A
CHAPTER 1: ENZYME-LINKED IMMUNOSORBENT ASSAY 1
(A review of Methodology)
1.1 ELISA systems 3
1.1.1 Non-competitive ELISA 3
1.1.1.1 Antigen coated plate 3
1.1.1.2 Antibody coated plate 3
1.1.2 Competitive ELISA 4
1.2 Choice of solid phase 4
1.3 Solid phase immobilization (coating of antigen or antibody) 5
1.3.1 Direct 5
1.3.2 Indirect 5
1.3.3 Concentration 6
1.3.4 Buffer and pH 6
1.3.5 Time 6
1.4 Interference 7
1.4.1 Blocking of unreacted sites 7
1.4.2 Heterophile antibodies 8
1.4.3 Other 8
1.4.4 Serum background 8
1.5 Washing 9
1.6 Samples, buffers and incubation times 10
1.7 Sample dilution and dispensing 12
1.8 Antibody reagents 12
1.8.1 Antibody reagents for IgG subclass ELISAs 13
1.9 Enzyme detector systems 14
1.10 Substrate 16
1.11 Plate format 17
1.12 Data acquisition 18
1.13 Date interpretation and quality control 22
CHAPTER 2: lgG SUBCLASS IMMUNOASSA YS 26
2.1 Introduction 27
2.2 Determination of optimal assay conditions 31
2.2.1 Coating monoclonal antibody 32
2.2.1.1 Selection 32
2.2.2 Backgrounds 32
2.2.2.1 IgG 1 subclass ELISA 33
2.2.2.2 IgG2 subclass ELISA 38
2.2.2.3 IgG3 subclass ELISA 39
2.2.2.4 IgG4 subclass ELISA 39
2.2.2.5 Conclusion and discussion 40
2.2.3 Coating buffer 45
2.2.3.1 IgGl subclass ELISA 45
2.2.3.2 IgG2 subclass ELISA 45
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2.2.3.3 IgG3 subclass ELISA 45
2.2.3.4 IgG4 subclass ELISA 46
2.2.3.5 Conclusion and discussion 48
2.2.4 Plates 49
2.2.4.1 IgG 1 subclass ELISA 49
2.2.4.2 IgG2 subclass ELISA 50
2.2.4.3 IgG3 subclass ELISA 50
2.2.4.4 IgG4 subclass ELISA 50
2.2.4.5 Conclusion and discussion 50
2.2.5 Blocking agents 51
2.2.5.1 IgG 1 subclass ELISA 51
2.2.5.2 IgG2 subclass ELISA 52
2.2.5.3 IgG3 subclass ELISA 53
2.2.5.4 IgG4 subclass ELISA 53
2.2.5.5 Conclusion and discussion 57
2.2.6 Serum diluents 58
2.2.6.1 IgG 1, IgG3 and IgG4 subclass ELISAs 58
2.2.6.2 IgG2 subclass ELISA 58
2.2.6.3 Conclusion and discussion 58
2.2.7 Serum standards 58
2.2.7.1 IgG 1 subclass ELISA 60
2.2.7.2 IgG2 subclass ELISA 61
2.2.7.3 IgG3 subclass ELISA 61
2.2. 7.4 IgG4 subclass ELISA 61
2.2.7.5 Conclusion and discussion 61
2.2.8 Serum incubation times 62
2.2.8.1 IgG 1, IgG2, IgG3 and IgG4 subclass ELISAs 62
2.2.8.2 Conclusion and discussion 63
2.2.9 Evaluation of the enzyme detection system 63
2.2.9.1 IgG 1 subclass ELISA 63
2.2.9.2 IgG2 subclass ELISA 65
2.2.9.3 IgG3 subclass ELISA 65
2.2.9.4 IgG4 subclass ELISA 65
2.2.9.5 Conclusion and discussion 66
2.3 Methods 67
2.3.1 IgG 1 subclass ELISA 67
2.3.2 IgG2 subclass ELISA 69
2.3.3 IgG3 subclass ELISA 69
2.3.4 IgG4 subclass ELISA 70
2.3.5 Design of plate 70
2.3.6 Calculation of results 71
2.4 Assay standardization 71
2.4.1 Standard curves 71
2.4.2 Precision and sensitivity 72
2.4.2.1 Coefficients of variation 72
2.4.2.2 Running means 72
2.4.2.3 Correlation with total IgG 74
2.4.2.4 Differences between the sum of the IgG subclasses 75
and the total IgG
2.4.3 Quality control 77
2.4.3.1 Inter-laboratory correlations 77
2.4.3.2 Quality assurance scheme 77
2.5 Summary 78
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CHAPTER 3: TETANUS TOXOID IMMUNOASSAYS 81
3.1 Introduction 82
3.2 Determination of optimal reagents and conditions 84
3.2.1 Plates 84
3.2.1.1 IgG tetanus toxoid ELISA 84
3.2.1.2 IgG 1 tetanus toxoid ELISA 85
3.2.1.3 IgG4 tetanus toxoid ELISA 86
3.2.1.4 Conclusion and discussion 87
3.2.2 Antigen coating 89
3.2.2.1 IgG tetanus toxoid ELISA 89
3.2.2.2 IgG 1 tetanus toxoid ELISA 90
3.2.2.3 IgG4 tetanus toxoid ELISA 90
3.2.2.4 Conclusion and discussion 90
3.2.3 Blocking agents 91
3.2.3.1 IgG tetanus toxoid ELISA 91
3.2.3.2 IgG 1 tetanus toxoid ELISA 93
3.2.3.3 IgG4 tetanus toxoid ELISA 93
3.2.3.4 Conclusion and discussion 93
3.2.4 Choice of serum standards and controls 93
3.2.4.1 IgG tetanus toxoid ELISA 94
3.2.4.2 IgG 1 tetanus toxoid ELISA 95
3.2.4.3 IgG4 tetanus toxoid ELISA 96
3.2.4.4 Conclusion and discussion 96
3.2.5 Serum incubation periods 96
3.2.6 Monoclonal antibody - selection 97
3.2.7 Monoclonal antibody - backgrounds 97
3.2.7.1 IgG 1 tetanus toxoid ELISA 101
3.2.7.2 IgG4 tetanus toxoid ELISA 102
3.2.7.3 IgG2 tetanus toxoid ELISA 103
3.2.7.4 IgG3 tetanus toxoid ELISA 104
3.2.7.5 Conclusion and discussion 104
3.2.8 Evaluation of enzyme detector systems 107
3.2.8.1 IgG tetanus toxoid ELISA 107
3.2.8.2 IgG 1 tetanus toxoid ELISA 107
3.2.8.3 IgG4 tetanus toxoid ELISA 109
3.2.8.4 Conclusion and discussion 109
3.3 Methods 109
3.3.1 IgG tetanus toxoid ELISA 109
3.3.2 IgG 1 tetanus toxoid ELISA 110
3.3.3 IgG4 tetanus toxoid ELISA 111
3.3.4 Design of plate 112
3.3.5 Calculation of results 113
3.3.6 Diethylamine-ELISA for IgG 1 and IgG4 tetanus toxoid 114
3.4 Assay standardisation 114
3.4.1 Standard curves 114
3.4.2 Precision and sensitivity 114
3.4.3 Specificity 116
3.5 Antibody affinity 119
3.5.1 Optimal dilution of DEA 119
3.5.2 Effect of DEA on solid-phase antigen 121
3.6 Summary 122
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CHAPTER 4: HAEMOPHILUS INFLUENZAE TYPE b IgG SUBCLASS 124
IMMUNOASSAY
4.1 Introduction 125
4.2 Determination of optimal reagents and conditions 127
4.2.1 Plates 127
4.2.1.1 Total IgG Hib PRP ELISA 127
4.2.1.2 IgG 1 Hib PRP ELISA 130
4.2.1.3 IgG2 Hib PRP ELISA 130
4.2.1.4 Conclusion and discussion 130
4.2.2 Comparison of PRP antigens 134
4.2.3 Antigen coating 136
4.2.3.1 IgG, IgG 1 and IgG2 Hib PRP ELIS As 136
4.2.3.2 Conclusion and discussion 137
4.2.4 Blocking agents and diluents 138
4.2.5 Serum standards and controls 139
4.2.5.1 IgG, IgG 1 and IgG2 Hib PRP ELIS As 139
4.2.6 Incubation period 141
4.2.7 Monoclonal antibody selection 141
4.2.7.1 IgG Hib PRP ELISA 141
4.2.7.2 IgG 1 Hib PRP ELISA 143
4.2.7.3 IgG2 Hib PRP ELISA 145
4.2.7.4 Conclusion and discussion 146
4.2.8 Evaluation of conjugated antibody/enzyme/substrates 146
4.2.8.1 IgG Hib PRP ELISA 146
4.2.8.2 IgG 1 Hib PRP ELISA 147
4.2.8.3 IgG2 Hib PRP ELISA 147
4.3 Methods 147
4.3.1 IgG Hib PRP ELISA 148
4.3.2 IgG 1 Hib PRP ELISA 149
4.3.3 IgG2 Hib PRP ELISA 149
4.3.4 Calculation of results 149
4.4 Assay standardization 150
4.4.1 Standard curves 150
4.4.2 Precision and sensitivity 152
4.4.3 Inter-laboratory comparison 152
4.4.4 Specificity 154
4.4.5 Antibody affinity 156
4.4.5.1 DEA-ELISA for IgGl and IgG2 Hib PRP 156
4.5 Summary 158
CHAPTER 5: STAPHYLOCOCCAL AUREUS lgG SUBCLASS 160
IMMUNOASSA YS
5.1 Introduction 161
5.2 Determination of optimal assay conditions 163
5.2.1 Plates 163
5.2.2 Coating antigen 163
5.2.2.1 Comparison of teichoic acid antigens 165
5.2.2.2 Comparison of commercial and in-house teichoic acid 165
antigen by RID
5.2.2.3 Comparison of commercial and in-house S. aureus teichoic 167
acid antigen by ELISA
5.2.2.4 Conclusion and discussion 167
5.2.3 Antigen coating 168
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5.2.3.1 IgG, IgG 1 and IgG2 teichoic acid ELIS As 168
5.2.4 Blocking agents 169
5.2.4.1 IgG, IgG 1 and IgG2 teichoic acid ELIS As 169
5.2.4.2 Conclusion and discussion 169
5.2.5 Serum standards and controls 170
5.2.5.1 IgG, IgG 1 and IgG2 teichoic acid ELIS As 170
5.2.6 Incubation periods 172
5.2.7 Monoclonal antibody - selection 172
5.2.7.1 IgG teichoic acid ELISA 172
5.2.7.2 IgG 1 teichoic acid ELISA 175
5.2.7.3 IgG2 teichoic acid ELISA 176
5.2.7.4 Conclusion and discussion 176
5.2.8 Evaluation of the enzyme detector system 177
5.2.8.1 IgG teichoic acid ELISA 177
5.2.8.2 IgG 1 teichoic acid ELISA 178
5.2.8.3 IgG2 teichoic acid ELISA 179
5.2.8.4 Conclusion and discussion 179
5.3 Methods 179
5.3.1 IgG teichoic acid ELISA 179
5.3.2 IgG 1 teichoic acid ELISA 180
5.3.3 IgG2 teichoic acid ELISA 181
5.3.4 Design of plate 182
5.3.5 Calculation of results 182
5.4 Assay standardization 183
5.4.1 Standard curves 183
5.4.2 Precision and sensitivity 184
5.4.3 Specificity 186
5.4.4 Antibody affinity 187
5.4.4.1 DEA-ELISA for IgGl and IgG2 teichoic acid 187
5.5 Summary 189
CHAPTER 6: G2m(n) and G lm(f) IMMUNOASSA YS 191
6.1. Introduction 192
6.2. Determination of optimal reagents and conditions 195
6.2.1 Plates 195
6.2.2 Coating - G2m(n) ELISA 195
6.2.2.1 Direct ELISA 195
6.2.2.2 Monoclonal capture ELISA 197
6.2.2.3 Conclusion and discussion 197
6.2.3 Coating - Glm(t) ELISA 199
6.2.3.1 Direct ELISA 199
6.2.3.2 Monoclonal capture ELISA 199
6.2.3.3 Conclusion and discussion 201
6.2.4 Antibody 202
6.2.4.1 G2m(n) ELISA 202
6.2.4.1.1 Polyclonal rabbit anti-G2m(n) antibody 202
6.2.4.1.2 Monoclonal anti-G2m(n) antibody 204
6.2.4.2 Glm(t) ELISA 205
6.2.4.5 Conclusion and discussion 205
6.2.5 Conjugates 206
6.2.5.1 G2m(n) ELISA 206
6.2.5.2 Glm(t) ELISA 207
6.3 Method 208
6.3.1 G2m(n) ELISA 208