Immunoassays: a practical approach
Gespeichert in:
| Weitere Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
New York, NY
Oxford University Press
2005
|
| Ausgabe: | Repr. |
| Schriftenreihe: | The practical approach series
|
| Schlagworte: | |
| Online-Zugang: | Klappentext Inhaltsverzeichnis |
| Beschreibung: | XIX, 304 S. Ill., graph. Darst. |
| ISBN: | 0199637105 |
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| adam_text | Immunoassays is the first practical manual designed to help any biologist develop an
immunoassay in any common format for any suitable analyte.
The book starts by describing the basic principles of immunoassay and by evaluating
the choice of assay types and formats. Following chapters describe, in full practical
detail: methods for raising and choosing polyclonal and monoclonal antibodies,
immunogen preparation and characterization, sourcing standards, the synthesis
and characterization of radioactive, enzymatic, fluorescent and ligand labels, the
preparation of a wide variety of solid-phase reagents, assay optimization (sensitivity
and accuracy), validation of quantitative and qualitative assays, curve fitting and
data processing (both manual and computer based), and quality assurance proce-
dures for small and large scale users.
This volume is unique in its scope and coverage of immunoassays. It is highly
recommended both for researchers new to immunoassays and those who are seek-
ing an updated source guide. The mix of background information, step by step
protocols, and strong practical advice for achieving success will prove invaluable
to any life scientist who chooses to use immunoassays in either a research
environment or for routine testing.
Cover illustration: Marcus Gosling
OXFORD
UNIVERSITY PRESS
ISBN 0-19-963710-5
78
199 6371
www.oup.com
Contents
Preface v
Protocol List xiii
Abbreviations xvii
1 Analysis by specific binding i
James P. Gosling
1 Introduction 1
2 Requirements for high-affinity specific binding 2
immunoglobulins 2
Antigen-antibody binding 5
3 The variety of immunoassays 7
Representing assay complexes in text 7
Classification 8
Nomenclature 23
The variety of formats 24
4 The limitations of binding assays 14
2 Raising antibodies 29
Marian M. Kane and John N. Banks
1 Introduction 19
Monoclonal and polyclonal antibodies 29
Antibodies and immunoassays 20
2 Animals and cell lines 21
Raising monoclonal antibodies 21
Raising polyclonal antibodies 23
3 Immunization 25
The immunogen 25
Adjuvants 26
Injection route and timing 29
4 Monitoring immunizations 32
General and when raising polyclonal antisera 32
When raising monoclonal antibodies 33
Screening procedures 33
CONTENTS
5 Making and manipulating hybridoma cells 37
Facilities and media 37
Fusions and selection of hybridomas 41
Cloning and further selections 45
Maintenance and storage 47
The production of monoclonal antibodies 49
6 Antibody purification 51
7 Emerging technologies 55
In vitro immunization 55
Recombinant antibody techniques 55
Choosing and characterizing antibodies 59
Anthony Forde and John Coley
1 Introduction 59
Secondary antibodies 59
Specificity 61
Epitope mapping 61
Affinity 62
2 Sourcing antibodies 67
3 Exploring specificity 69
Specificity as assessed by suppliers 69
Specificity and the assay developer 70
4 Estimating affinity 76
Approaches 76
Methods 77
5 Conclusion 86
4 Labels and endpoints 89
Wajdi Abdul-Ahad and Martin Brett
1 Labelling substances 89
Radioisotopic labels 89
Enzyme labels 92
Fluorescent labels 93
Ligands and indirect labelling 94
2 Enzyme measurement methods 95
Methods with chromogens 95
Methods with fluorogens 97
Methods that generate luminescence 98
3 Labelling and conjugation 99
Radiolabelling 99
The chemistry of conjugation 104
Protein-protein conjugation 109
Enzyme hapten coupling 117
Fluorescence labelling 121
Biotin coupling 122
4 Label characterization 123
5 Conjugate stabilization 125
viii
S Solid-phase reagents 129
Robert S. Matson
1 Introduction 129
Liquid-phase assay with solid-phase separation 130
Choice of solid-phase support 130
2 Approaches to immobilization 131
Preparation of ligands for immobilization 132
Covalent attachment 132
Noncovalent adsorption 140
Indirect immobilization 141
Treatments after coating 142
3 Solid-phase formats 144
Microtitre plates 146
Tubes 150
Membranes 150
Microspheres 155
4 Handling and storage 160
Surface dénaturation 160
Microtitre wells and tubes 160
Membranes 161
Micro spheres 161
5 Sourcing of reagents 161
6 Standards and Immunogens 165
M. Quasim Chaudry
1 Introduction 165
Criteria for a good immunogen 165
Animal welfare 167
2 Purification of immunogens 168
3 Synthetic and recombinant immunogens 169
Synthetic peptide immunogens 169
Recombinant peptide immunogens 170
4 Conjugation of haptens and carriers 170
Materials and reagents 171
Methods 172
Characterization of hapten immunogens 180
5. Storage of immunogens 182
6. Standards 182
7 Optimization 187
Eric Ezan and Jacques Grassi
1 Introduction 187
2 Sensitivity 187
Estimators of sensitivity 187
Optimization of reagent concentrations 191
Optimization of physiochemical parameters 194
CONTENTS
3 Accuracy 196
Factors affecting accuracy 196
Technological variability 298
Crossreactive compounds 199
Plasma-binding proteins 200
Anti-Ig antibodies 203
4 Conclusions 208
Validation 211
Peter O Fegan
1 Introduction 211
Validation in the development cycle 211
Validation criteria and analytical goals 212
2 Specificity 212
Crossreactivity studies 213
3 Precision 217
Materials 217
Levels of precision 218
4 Sensitivity 222
5 Accuracy 224
Recovery of added standard 224
Independence of volume 226
Comparison with a reference method 228
6 Biological validation 230
Concentrations and changes in concentration 230
Normal ranges and diagnostic cut-off levels 231
7 Qualitative and semi-quantitative tests 232
Specificity 232
Validation criteria 233
8 Evaluation of reagent stability 235
9 Data processing 239
Barry Nix and David Wild
1 Introduction 239
2 Fitting calibration curves 240
Definitions 240
Manual and computer methods 241
Computer methods in common use 245
Evaluating candidate methods 247
3 More worked examples 254
A good logit-log fit 254
Consequences of a poor logit-log fit 254
4 Calibration curve-fit software 260
X
CONTENTS
10 Quality assurance 263
John Seth
1 Introduction 263
2 Quality assurance 263
Principles 263
Specimen collection and preservation 264
Reporting results 265
3 Internal quality control 265
Definitions 265
Pre-analytical quality control 266
Quality control of the analysis 268
Post analytical quality control 282
Limitation of IQC 282
4 External quality assessment 282
Principles 282
Uses and availability of EQA schemes 283
Operation of EQA schemes for immunoassay 285
Limitations of EQA 289
Making use of EQA and IQC data 292
A1 Suitable reagents and other requisites
Index 299
XI
|
| adam_txt |
Immunoassays is the first practical manual designed to help any biologist develop an
immunoassay in any common format for any suitable analyte.
The book starts by describing the basic principles of immunoassay and by evaluating
the choice of assay types and formats. Following chapters describe, in full practical
detail: methods for raising and choosing polyclonal and monoclonal antibodies,
immunogen preparation and characterization, sourcing standards, the synthesis
and characterization of radioactive, enzymatic, fluorescent and ligand labels, the
preparation of a wide variety of solid-phase reagents, assay optimization (sensitivity
and accuracy), validation of quantitative and qualitative assays, curve fitting and
data processing (both manual and computer based), and quality assurance proce-
dures for small and large scale users.
This volume is unique in its scope and coverage of immunoassays. It is highly
recommended both for researchers new to immunoassays and those who are seek-
ing an updated source guide. The mix of background information, step by step
protocols, and strong practical advice for achieving success will prove invaluable
to any life scientist who chooses to use immunoassays in either a research
environment or for routine testing.
Cover illustration: Marcus Gosling
OXFORD
UNIVERSITY PRESS
ISBN 0-19-963710-5
78
199 6371
www.oup.com
Contents
Preface v
Protocol List xiii
Abbreviations xvii
1 Analysis by specific binding i
James P. Gosling
1 Introduction 1
2 Requirements for high-affinity specific binding 2
immunoglobulins 2
Antigen-antibody binding 5
3 The variety of immunoassays 7
Representing assay complexes in text 7
Classification 8
Nomenclature 23
The variety of formats 24
4 The limitations of binding assays 14
2 Raising antibodies 29
Marian M. Kane and John N. Banks
1 Introduction 19
Monoclonal and polyclonal antibodies 29
Antibodies and immunoassays 20
2 Animals and cell lines 21
Raising monoclonal antibodies 21
Raising polyclonal antibodies 23
3 Immunization 25
The immunogen 25
Adjuvants 26
Injection route and timing 29
4 Monitoring immunizations 32
General and when raising polyclonal antisera 32
When raising monoclonal antibodies 33
Screening procedures 33
CONTENTS
5 Making and manipulating hybridoma cells 37
Facilities and media 37
Fusions and selection of hybridomas 41
Cloning and further selections 45
Maintenance and storage 47
The production of monoclonal antibodies 49
6 Antibody purification 51
7 Emerging technologies 55
In vitro immunization 55
Recombinant antibody techniques 55
Choosing and characterizing antibodies 59
Anthony Forde and John Coley
1 Introduction 59
Secondary antibodies 59
Specificity 61
Epitope mapping 61
Affinity 62
2 Sourcing antibodies 67
3 Exploring specificity 69
Specificity as assessed by suppliers 69
Specificity and the assay developer 70
4 Estimating affinity 76
Approaches 76
Methods 77
5 Conclusion 86
4 Labels and endpoints 89
Wajdi Abdul-Ahad and Martin Brett
1 Labelling substances 89
Radioisotopic labels 89
Enzyme labels 92
Fluorescent labels 93
Ligands and indirect labelling 94
2 Enzyme measurement methods 95
Methods with chromogens 95
Methods with fluorogens 97
Methods that generate luminescence 98
3 Labelling and conjugation 99
Radiolabelling 99
The chemistry of conjugation 104
Protein-protein conjugation 109
Enzyme hapten coupling 117
Fluorescence labelling 121
Biotin coupling 122
4 Label characterization 123
5 Conjugate stabilization 125
viii
S Solid-phase reagents 129
Robert S. Matson
1 Introduction 129
Liquid-phase assay with solid-phase separation 130
Choice of solid-phase support 130
2 Approaches to immobilization 131
Preparation of ligands for immobilization 132
Covalent attachment 132
Noncovalent adsorption 140
Indirect immobilization 141
Treatments after coating 142
3 Solid-phase formats 144
Microtitre plates 146
Tubes 150
Membranes 150
Microspheres 155
4 Handling and storage 160
Surface dénaturation 160
Microtitre wells and tubes 160
Membranes 161
Micro spheres 161
5 Sourcing of reagents 161
6 Standards and Immunogens 165
M. Quasim Chaudry
1 Introduction 165
Criteria for a good immunogen 165
Animal welfare 167
2 Purification of immunogens 168
3 Synthetic and recombinant immunogens 169
Synthetic peptide immunogens 169
Recombinant peptide immunogens 170
4 Conjugation of haptens and carriers 170
Materials and reagents 171
Methods 172
Characterization of hapten immunogens 180
5. Storage of immunogens 182
6. Standards 182
7 Optimization 187
Eric Ezan and Jacques Grassi
1 Introduction 187
2 Sensitivity 187
Estimators of sensitivity 187
Optimization of reagent concentrations 191
Optimization of physiochemical parameters 194
CONTENTS
3 Accuracy 196
Factors affecting accuracy 196
Technological variability 298
Crossreactive compounds 199
Plasma-binding proteins 200
Anti-Ig antibodies 203
4 Conclusions 208
Validation 211
Peter O'Fegan
1 Introduction 211
Validation in the development cycle 211
Validation criteria and analytical goals 212
2 Specificity 212
Crossreactivity studies 213
3 Precision 217
Materials 217
Levels of precision 218
4 Sensitivity 222
5 Accuracy 224
Recovery of added standard 224
Independence of volume 226
Comparison with a reference method 228
6 Biological validation 230
Concentrations and changes in concentration 230
Normal ranges and diagnostic cut-off levels 231
7 Qualitative and semi-quantitative tests 232
Specificity 232
Validation criteria 233
8 Evaluation of reagent stability 235
9 Data processing 239
Barry Nix and David Wild
1 Introduction 239
2 Fitting calibration curves 240
Definitions 240
Manual and computer methods 241
Computer methods in common use 245
Evaluating candidate methods 247
3 More worked examples 254
A good logit-log fit 254
Consequences of a poor logit-log fit 254
4 Calibration curve-fit software 260
X
CONTENTS
10 Quality assurance 263
John Seth
1 Introduction 263
2 Quality assurance 263
Principles 263
Specimen collection and preservation 264
Reporting results 265
3 Internal quality control 265
Definitions 265
Pre-analytical quality control 266
Quality control of the analysis 268
Post analytical quality control 282
Limitation of IQC 282
4 External quality assessment 282
Principles 282
Uses and availability of EQA schemes 283
Operation of EQA schemes for immunoassay 285
Limitations of EQA 289
Making use of EQA and IQC data 292
A1 Suitable reagents and other requisites
Index 299
XI |
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| spelling | Immunoassays a practical approach ed. by James P. Gosling Repr. New York, NY Oxford University Press 2005 XIX, 304 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series Methode (DE-588)4038971-6 gnd rswk-swf Immunassay (DE-588)4113998-7 gnd rswk-swf Immunassay (DE-588)4113998-7 s DE-604 Methode (DE-588)4038971-6 s Gosling, James Patrick (DE-588)1202355064 edt Digitalisierung UB Bayreuth - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015660321&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Klappentext Digitalisierung UB Bayreuth - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015660321&sequence=000002&line_number=0002&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Immunoassays a practical approach Methode (DE-588)4038971-6 gnd Immunassay (DE-588)4113998-7 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4113998-7 |
| title | Immunoassays a practical approach |
| title_auth | Immunoassays a practical approach |
| title_exact_search | Immunoassays a practical approach |
| title_exact_search_txtP | Immunoassays a practical approach |
| title_full | Immunoassays a practical approach ed. by James P. Gosling |
| title_fullStr | Immunoassays a practical approach ed. by James P. Gosling |
| title_full_unstemmed | Immunoassays a practical approach ed. by James P. Gosling |
| title_short | Immunoassays |
| title_sort | immunoassays a practical approach |
| title_sub | a practical approach |
| topic | Methode (DE-588)4038971-6 gnd Immunassay (DE-588)4113998-7 gnd |
| topic_facet | Methode Immunassay |
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