Immunoassay

Front Cover

Immunoassay

Copyright Page

Contents

Contributors

Preface

CHAPTER 1. PAST, PRESENT AND FUTURE OF IMMUNOASSAYS

CHAPTER 2. IMMUNE FUNCTION AND ANTIBODY STRUCTURE

1. Cellular and Molecular Basis of the Humoral Immune Response

2. Antibody Structure

3. Antigen Binding

4. Antibody Structure and Immunologic Diversity

5. Conclusion

CHAPTER 3. THEORY OF IMMUNOASSAYS

1. Binding Theory

2. Equilibria Involving Many Ligands and a Heterogeneous Population of Binding Sites

3. Principles of Saturation Analysis

4. Kinetics of Immunoassay

5. The Theory of Two-Site Immunoassays

6. Graphical Presentation of Immunoassay Data

7. Curve Fitting of Immunoassay Data

8. Detection Limit of Immunoassays

References

CHAPTER 4. DATA INTERPRETATION AND QUALITY CONTROL

1. Introduction

2. Assay Validation

3. Determining Immunoassay Sensitivity

4. Tests of Similarity (Parallelism)

5. Alternative Methods for Defining Cross Reactivity

6. Quality Control

7. Summary

References

CHAPTER 5. PRODUCTION AND PURIFICATION OF ANTIBODIES

1. Introduction

2. Polyclonal versus Monoclonal Antibodies

3. Choice of Animal

4. Immunization Procedures

5. Obtaining and Processing Blood and Lymphocytes

6. Monoclonal Antibody Production

7. Antibody Purification

References

CHAPTER 6. ANTIBODY ENGINEERING

1. Introduction

2. Cloning Antibody Variable Region Genes

3. Expression of Recombinant Antibodies

4. Antibody Structure-Function Studies

5. Therapeutic Antibodies

6. Designer Antibodies

7. Future Directions

References

CHAPTER 7. INTERFERENCES IN IMMUNOASSAYS

1. Introduction

2. Detecting Interferences

3. Cross-Reactivity and Heterogeneous Analytes

4. Reducing Cross-Reactive Interferences

5. Heterophile and Anti-Animal Antibodies

6. Recognizing and Reducing Interference from Endogenous Antibodies

7. Interferences Due to Masking of Antigens

8. Interference with the Indicator Mechanism

9. Matrix Effects

10. Interference Problems in the Future

References

CHAPTER 8. LABELING OF ANTIBODIES AND ANTIGENS

1. Introduction

2. Requirements for Quality of Labeled Reactants

3. Chemical Reactions for Conjugation

4. Reagents for Conjugation

5. Conjugation of Antibody IgG to Enzymes

6. Conjugation of Antibody Fragments to Enzymes

7. Conjugation of Antigens, Avidin, and Streptavidin to Enzymes

8. Conjugation of Small Molecules to Antibodies and Antigens

References

CHAPTER 9. SOLID PHASES IN IMMUNOASSAY

1. Interfacial Immunochemistry

2. Diversity of Solid Phases

3. Immobilization Procedures

4. SPI in the Future

References

CHAPTER 10. IMMUNOASSAY CONFIGURATIONS

1. Introduction

2. Competitive Immunoassays

3. Noncompetitive Immunoassays

4. Indirect Detection of the Immunocomplexes

References

CHAPTER 11. THE AVIDIN-BIOTIN SYSTEM

1. Introduction

2. Components of the System

3. Amplified Detection Systems

4. Improvement of Capture Systems

5. Auxiliary Enhancement Systems

6. Selected Protocols

References

CHAPTER 12. RADIOIMMUNOASSAY

1. Radioactive Isotopes

2. Counting of Radioactive Isotopes

3. Characteristics of a Tracer

4. Preparation of Tracers

5. Iodinated Tracers

6. Iodination Methods

7. Iodination Damage

8. Comparison of Labeled and Unlabeled Ligand

9. Purification of Iodinated Tracer

10. Chemical Evaluation of Tracer

11. Immunological Evaluation of Tracer

12. Variations on the Use of Radiolabeled Tracers

Reference

CHAPTER 13. ENZYME IMMUNOASSAY

1. Introduction

2. Enzymes as Labeling Substances

3. Conjugation Procedures

4. Determining Enzyme Activity

5. The Variety of Enzyme Immunoassays

6. Prospects

References

CHAPTER 14. FLUORESCENCE IMMUNOASSAYS

1. Introduction

2. Principles of Fluorescence Spectroscopy

3. Fluorescence Immunoassay Instrumentation

4. Fluorescent Labels

5. Homogeneous Fluorescence Immunoassays

6. Heterogeneous Fluorescence Immunoassays

References

CHAPTER 15. CHEMILUMINESCENCE IMMUNOASSAY

1. Introduction

2. Chemiluminescent Labels and Labeling

3. Detection of Labels

4. Types of Chemiluminescent Immunoassay

5. Chemiluminescent Detection of Enzyme Labels

6. Automated Chemiluminescent Immunoassay Systems

References

CHAPTER 16. BIOLUMINESCENCE IMMUNOASSAYS

1. Introduction

2. Principles of Bioluminescent Light Emission

3. Bioluminescence Immunoassay Instrumentation

4. Enzyme Labels and Bioluminogenic Substrates

5. Bioluminescence Enzyme Immunoassays

6. Other Bioluminescence Immunoassay Systems

7. Availability of Bioluminescence Immunoassays

References

CHAPTER 17. NEPHELOMETRIC AND TURBIDIMETRIC IMMUNOASSAY

1. Introduction

2. Historical Perspective

3. Characteristics of the Immunoprecipitin Reaction

4. Principles of Light Scattering

5. Definition and Principles of Nephelometry and Turbidimetry

6. Instrumentation

References

CHAPTER 18. SIMULTANEOUS MULTIANALYTE IMMUNOASSAYS

1. Introduction

2. Labels and Immunoassay Formats

3. Labels

4. Discrete Test Zones

5. Test Combinations

6. Conclusions

References

CHAPTER 19. NONCOMPETITIVE IMMUNOASSAY FOR SMALL MOLECULES

1. Introduction

2. Anti-idiotypic Antibodies as Novel Probes in Immunodiagnostics

3. Idiometric Assays for Estradiol

4. Idiometric Assays for Progesterone

5. Conclusions

References

CHAPTER 20. FREE HORMONE MEASUREMENTS

1. Introduction

2. Overview of Thyroid, Steroid, and Secosteroid Hormones

3. Transport of Thyroid, Steroid, and Secosteroid Hormones in the Blood

4. The Theory of Free Hormones and Alternative Concepts

5. History of Free Hormone Assays

6. Classification of Free Hormone Assays

7. Principles of Free Hormone Assays

8. Total Hormone/Total Hormone-Binding Globulin Ratio

9. Tests Based on the Measurement of "Thyroid Hormone Binding"

10. Calculated Free Hormone Concentrations

11. Non-Sex Hormone-Binding Globulin-Bound Testosterone (NSB-Te)

12. Assay Parameters of Free Hormone Assays

13. Specimens for Free Hormone Assays

14. Interferences with Free Hormone Measurements

15. Selected Issues Regarding Interpretation of Free Hormone Results

16. Analytical Goals for Free Hormone Measurements

17. Quality Control of Free Hormone Assays

18. Conclusions and Perspectives of Free Hormone Measurements

References

CHAPTER 21. AUTOMATION OF IMMUNOASSAYS

1. Introduction

2. Automation of Immunoassay

3. Automated Immunoassay Systems

4. Performance of Immunoassay Systems

5. Future Trends

References

CHAPTER 22. THIN-FILM IMMUNOASSAYS

1. Introduction

2. Basic Features of Dry Reagent Assays

3. Examples of Thin-Film Immunoassays

4. Conclusion

References

CHAPTER 23. IMMUNOBLOTTING TECHNIQUES

1. Introduction

2. Principles

3. The Basic Protocol

4. Detection Methods

5. Specialized Uses and Applications

6. Conclusions

Reference

CHAPTER 24. DEVELOPMENT OF IN-HOUSE IMMUNOLOGICAL ASSAYS

1. Introduction

2. Development of an Immunoassay Method

3. A Practical Example--An ELISA for p53

4. Conclusions

References

INDEX