Immunodiagnostics : a practical approach

Abbreviations xvii 1. Principles of immunodiagnostic tests and their development; with specific use of radioisotopes as tracers Raymond Edwards, Stuart Blincko and Iain Howes 1. Introduction 1 2. Classification of type 3 3. Selection and preparation of reagents 5 Antibody or antiserum 6 Selection of antibody or antiserum 12 Purification of antibodies and antisera 17 Selection of label 27 Preparation of 125I-radioiodinated tracers 27 Purification of 125I-radioiodinated tracers 30 Selection and preparation of standard or calibrant 31 4. Assay design 34 Assay format 35 General reagents and additives 38 Sample preparation 39 Optimal protocol and concentration of reagents 41 Assay validation 43 Appendix 46 References 48 2. Solid-phase supports 51 Stuart Blincko, Shen Rongsen, Shen Decun, Iain Howes and Raymond Edwards 1. Introduction 51 2. Solid phase supports 54 Polystyrene 54 Other organic polymers 54 Cellulose particles 54 Agarose, sephacryl and sephadex particles 55 1 Contents Magnetizable particles 55 Membranes 56 3. Preparation of solid supports 56 4. Passive adsorption 58 5. Covalent coupling 61 Derivatization of solid supports 61 Covalent coupling reactions with solid supports 64 6. Indirect coupling methods 88 Indirect coupling of antibody via a second antibody 88 Indirect coupling of antibody via binding proteins (A and G) 91 Indirect binding of antibody via the biotin-avidin interaction 92 Indirect binding of antibodies labelled with a hapten (fluorescein) 94 7. Blocking 94 8. Storage 95 9. Assay wash solutions 95 Appendix A: Suppliers of solid supports 96 Appendix B: List of suppliers 97 References 97 3. Enzyme-labelled tests with colorimetric, fluorimetric and chemiluminescent detection systems 101 Iain Howes, Stuart Blincko, John Little and Raymond Edwards 1. Introduction 101 2. Selection of enzyme-labels 101 3. Enzyme preparation and assignment of enzymatic activity 102 Preparations 102 Enzymatic assays 104 4. Conjugation methods 108 Activation of small molecules and their covalent coupling to enzymes 108 Activation of glycoprotein enzymes and their coupling to proteins 109 Activation of glycoproteins and their covalent coupling to enzymes 110 Coupling via bifunctional linking molecules 110 5. Purification, storage and testing of enzyme conjugates 115 Gel filtration of conjugate 115 Lectin chromatography of glycoprotein enzyme conjugates 116 Fractionation of antibody conjugates by Protein A chromatography 117 Stabilization and storage of enzyme conjugates 119 Testing of conjugates 119 Contents 6. Signal generation 121 Spectrophotometric systems for solid phase enzyme-labelled assays 122 Enzyme amplification 125 Immunostaining procedures 125 Fluorescent systems 125 Enhanced chemiluminescent systems 126 References 131 4. Time-resolved fluorescence immunoassay 137 Geoff Barnard 1. Introduction 137 2. Principles of time-resolved fluorescence 137 3. Protein labelling with lanthanide chelates 140 4. Storage of labelled proteins 143 5. Application to two-site immunometric assay of proteins 143 6. Application to competitive binding immunoassays of haptens 146 7. Europium-labelled hapten assay 148 8. Monoclonal antibody screening procedures 149 9. Novel research assays 151 10. Non-separation time-resolved fluoroimmunoassay 151 11. Simultaneous immunoassays 152 12. Conclusion 156 Acknowledgements 157 Appendix: List of suppliers 157 References 158 5. Light scattering techniques 159 David J. Newman, Hansa Thakkar and Christopher P. Price 1. Introduction 159 2. Fundamental aspects of immunoaggregation 159 Monitoring techniques 161 End-point versus rate measurement 164 Analyte and assay format 164 Antigen excess 165 Optimization strategies 166 Validation strategies 166 xi Contents 3. Non-enhanced methodology 167 Antibody selection 170 Choice of buffer and PEG concentration 172 Assay performance 174 4. Enhanced methodology 174 Choice of assay format 178 Selection of particle 178 Coupling chemistry 181 Evaluation of particle reagents 186 Antibody selection and optimization 188 Buffer optimization 189 Assay performance 192 5. Summary 194 References 195 Acknowledgements 196 6. Enzyme amplification: A means to develop fast ultrasensitive imimmoassays 197 Colin H. Self, David Bates and David B. Cook 1. Introduction 197 2. Practical applications 200 Water and buffers 200 Colorimetric enzyme amplification 201 3. Fluorimetric enzyme amplification 206 4. Conclusions 211 Appendix: List of suppliers 212 References 213 7. Equipment and automation; Appendix on dose-response curve fitting 215 Iain Howes and Raymond Edwards 1. Introduction 215 Features of automation 215 2. Steps in immunoassay analysis 216 Sample identification—bar-coding 216 Worklist generation 216 Transfer of samples to reaction vessel 216 Addition of immunoreagents 217 Incubation 217 xii Contents Separation where necessary 217 Signal generation 217 Signal detection 217 Curve fitting—response calibration 217 Quality control 218 Report generation 218 Export of results to LIMS 218 3. Equipment 219 Bar code readers 219 Worklist generators 219 Pippettors/diluters 219 Reagent dispensers 219 Incubators/shakers 221 Washers 221 Signal detectors 221 Response curve calibration software 222 Quality control 222 Sample/reagent processors 222 Assay processors 223 Robotic arms 227 Fully automated analysers 227 Clinical chemistry analysers 238 Cleaning and disinfection 238 Safety features 239 4. Analytical considerations 239 5. Conclusions 239 References 240 Appendix 240 8. Quality assurance in immunodiagnostics 243 David G. Bullock and Finlay MacKenzie 1. Introduction 243 The need for quality 243 The purpose of quality assurance 243 Implementation of a quality assurance programme 244 2. Quality systems 244 Quality assurance (QA) 244 Internal quality control (IQC) 245 External quality assessment (EQA) 245 Audit 245 Accreditation of laboratories 245 Validation of results 246 Good manufacturing practice (GMP) 246 xiii Contents Training and education 247 Evaluation of reagents and equipment 247 3. Quality assurance 247 Test selection and siting 248 Pre-analytical quality assurance 248 Post-analytical quality assurance 249 Non-analytical quality assurance 249 Summary 250 4. Internal quality control 250 Simple IQC procedures 251 Statistical quality control techniques 251 Validated control techniques 252 Techniques using patients' data 253 Assay validation 253 Clinical validation 257 Corrective action 257 Summary 258 5. External quality assessment 258 The purpose of EQA 258 Practical considerations 259 Scheme design 259 Competition in EQA 263 How to use EQA participation 264 Selection of an EQA scheme 264 6. Materials for IQC and EQA 265 Material sources and preparation 265 Material presentation 266 Summary 267 7. Discussion 267 Why use QA at all? 267 What is good enough? 267 What reference intervals should be used? 268 Who has responsibility for quality? 268 Future prospects 268 Acknowledgements 269 References 269 List of suppliers 271 Index 277