Immunoassay: A Practical Guide (Ellis Horwood Series in Pharmaceutical Technology)

Book Cover......Page 1
Title......Page 3
Copyright......Page 4
Contents......Page 5
List of Contributors......Page 6
Preface......Page 8
1 Introduction......Page 9
References......Page 13
Chemical hazards......Page 14
Radiological hazards......Page 15
Iodine-125......Page 16
Monitoring......Page 17
Bibliography......Page 18
Introduction......Page 19
Point of attachment......Page 20
Functionalisation of the hapten......Page 22
Carrier protein......Page 24
Immunogen incorporation ratios......Page 25
Carbodiimide reaction (−COOH + NH2−)......Page 27
N-Hydroxysuccinimide ester mediated conjugation (−COOH+NH2−)......Page 29
Mixed anhydride procedure (−COOH+NH2−)......Page 30
Glutaraldehyde condensation (−NH2+NH2−)......Page 31
DFDNB (−NH2 + NH2−)......Page 32
Other reactions......Page 33
Purification of conjugates......Page 34
Radiolabelled methods......Page 35
Other methods......Page 36
Storage of immunogens......Page 37
References......Page 38
Introduction......Page 40
Heavy chains......Page 41
Fragments of IgG antibodies......Page 42
Variable regions and antigen binding......Page 44
Affinity, avidity, titre and specificity......Page 46
Genetic structure of the germ line......Page 48
Clonal selection......Page 49
Control mechanisms......Page 50
Somatic mutation and affinity maturation......Page 51
Tolerance......Page 54
Polyclonal immune serum or monoclonal antibody?......Page 55
Choice of species......Page 56
Sheep......Page 57
Number of animals......Page 58
Adjuvants......Page 59
Preparation of emulsions......Page 60
Immunisation schedules......Page 62
Blood withdrawal......Page 63
Recommended immunisation procedures......Page 65
Storage and stability of antiserum......Page 68
Molecular biology......Page 69
References......Page 70
Introduction......Page 72
Tritium......Page 73
Radioactive decay processes......Page 74
14C labelling......Page 75
3H labelling (tritiation)......Page 76
Direct iodination......Page 77
Bolton-Hunter reagent......Page 81
Iodohistamine......Page 84
Bridge recognition......Page 88
High-performance liquid chromatography (HPLC)......Page 92
Thin-layer chromatography (TLC)......Page 93
Iodinated tracers......Page 94
Solvent extraction......Page 95
High-performance liquid chromatography (HPLC)......Page 96
Tritiated tracers......Page 99
Tracer purity......Page 101
Stability of radiotracers......Page 102
The effect of storage solvent......Page 103
Effect of storage container......Page 104
Conclusions......Page 105
References......Page 106
Introduction......Page 108
Assay tubes......Page 109
Aspirator......Page 110
Tracer mass......Page 112
Buffers and buffer additives......Page 113
Separating systems......Page 114
Ammonium sulphate......Page 118
Polyethylene glycol 6000 (PEG)......Page 119
Charcoal......Page 120
Assay conditions......Page 122
Assessment of antibodies......Page 123
Introduction of matrix......Page 127
Assay optimisation......Page 129
Separation methods......Page 130
Second antibody methods......Page 131
Scintillation proximity assays (SPA)......Page 132
Optimising for sensitivity......Page 133
Optimising for specificity......Page 135
Optimising for precision......Page 136
Optimising for accuracy......Page 137
References......Page 138
Introduction......Page 141
Assay buffers......Page 144
Substrate development buffers......Page 146
Microtitre plates......Page 147
Washing apparatus......Page 148
Automated systems......Page 149
Carrier proteins......Page 150
Bridge recognition......Page 151
Defining the starting conditions......Page 152
Antiserum assessment......Page 153
Optimisation of the sensitisation stage......Page 155
Optimisation of non-specific binding......Page 157
Optimisation of the primary antibody incubation......Page 158
Introduction of matrix......Page 159
Optimisation of the second antibody incubation......Page 160
Precision......Page 161
Convenience......Page 162
Conclusions......Page 163
References......Page 164
Introduction......Page 165
Standard curves from a simple analogy for radioimmunoassay......Page 166
Ideal systems......Page 168
The effect of non-ideal aspects of immunoassay......Page 171
Modelling the shape—logistic models for immunoassay calibration plots......Page 172
Linear (logit/log) calibration plots......Page 173
Non-linear logistic calibration plots......Page 175
Modelling the data—empirical models for immunoassay calibration plots......Page 179
Errors in fitting immunoassay calibration plots......Page 181
Comparison of approaches to fitting immunoassay calibration plots......Page 182
Replication of standards......Page 183
Response measurement......Page 184
References......Page 185
Introduction......Page 187
Response function......Page 188
Precision......Page 189
Accuracy......Page 190
Limits of quantification......Page 192
Limit of detection or sensitivity......Page 193
Specificity......Page 195
Stability......Page 197
Experimental plan......Page 199
References......Page 200
The aims of a QC policy......Page 201
Precision, bias and accuracy......Page 202
Response error relationships and the precision profile......Page 203
Limits of quantification......Page 206
Within and between batch error......Page 207
QC rules and their ‘power functions’......Page 208
Quality control samples......Page 209
Relationship of QC rules to assay quality and assay stability......Page 212
Recommendations......Page 215
Critical error......Page 216
Interpretation......Page 217
Appendix 1: The use of one-way analysis of variance to determine within and between batch error......Page 218
References......Page 219
Introduction......Page 221
Problems with standards......Page 222
Contamination problems......Page 224
Poor precision......Page 225
Difficulties in transferring an assay to another laboratory......Page 228
Conclusions......Page 229
Index......Page 234