Enzyme-linked Immunosorbent Assay (ELISA) - From A to Z

Preface

Acknowledgements

Contents

1 Fundamentals and History of ELISA: The Evolution of the Immunoassays Until Invention of ELISA

Abstract

1.1 Evolution of the Immunoassays Until Invention of ELISA

1.1.1 Side Chain Theory

1.1.2 Antigen-Antibody Binding Theory

1.1.3 Discovery of Antibody Structure

1.1.4 Invention of Radioimmunoassay (RIA)

1.1.5 Invention of Enzyme Linked Immunosorbent Assay (ELISA)

1.2 Principles of the Immune System

1.2.1 Antibody Production in Human Body

1.2.2 Different Types of Antibodies

1.2.2.1 Immunoglobulin G (IgG)

1.2.2.2 Immunoglobulin A (IgA)

1.2.2.3 Immunoglobulin M (IgM)

1.2.2.4 Immunoglobulin D (IgD)

1.2.2.5 Immunoglobulin E (IgE)

1.2.3 Antigen-Antibody Coupling

1.2.3.1 Specificity of the Antigen-Antibody Coupling

1.3 Biomolecular Interactions Between Antibody and Antigen

1.3.1 Hydrogen Bonding

1.3.2 Hydrophobic Interaction

1.3.3 Ionic Attraction

1.3.4 Van der Waals Forces

1.3.4.1 London Dispersion Force

1.3.4.2 Dipole-Dipole Interaction

1.3.4.3 Ion-Dipole Interaction

References

2 General Overviews on Applications of ELISA

Abstract

2.1 Applications of ELISA

2.1.1 Food Industry

2.1.2 Vaccine Development

2.1.3 Immunology

2.1.3.1 Autoimmunity

2.1.3.2 Humoral Immunity

2.1.4 Diagnosis

2.1.4.1 Pregnancy Test

2.1.4.2 Cancer Detection

2.1.4.3 Detection of the Infectious Diseases

2.1.5 Toxicology

2.1.6 Drug Monitoring and Pharmaceutical Industry

2.1.7 Transplantation

References

3 Step by Step with ELISA: Mechanism of Operation, Crucial Elements, Different Protocols, and Insights on Immobilization and Detection of Various Biomolecular Entities

Abstract

3.1 Mechanism of Operation

3.2 Different Elements of the Assay

3.2.1 Solid Phase

3.2.2 Adsorbents

3.2.2.1 Target Biomolecules

3.2.3 Washing Agents

3.2.4 Blocking Agents

3.2.5 Enzymes and Substrates

3.2.5.1 Different Types of Enzyme

3.2.5.2 Different Types of Substrate

3.2.6 Stopping Process

3.2.7 Reading Techniques

3.2.7.1 Colorimetric Assay

3.2.7.2 Fluorescent Assay

3.2.7.3 Luminescent Assay

3.2.8 Reading Apparatus

3.2.9 Controls

3.2.9.1 Positive Controls

3.2.9.2 Endogenous Positive Control

3.2.9.3 Negative Controls

3.2.9.4 Standard Controls

3.2.9.5 Spike Controls

3.3 Different Protocols

3.3.1 Direct ELISA

3.3.2 Indirect ELISA

3.3.3 Sandwich ELISA

3.3.4 Double Sandwich ELISA

3.3.5 Competitive ELISA

3.4 Initial Interaction of the Biomolecules with the Surface

3.5 Immobilization Techniques for Protein Attachment

3.5.1 Physical Immobilization

3.5.2 Immobilization via Entrapment

3.5.3 Covalent Immobilization

3.5.3.1 Immobilization via Zero-Length Cross Linker

3.5.3.2 Immobilization via Spacers

3.5.4 Oriented Immobilization

References

4 Evaluation of the Detection Results Obtained from ELISA

Abstract

4.1 Conducting a Reliable Assay

4.1.1 Sources of Errors

4.1.2 Troubleshooting

4.2 Key Parameters in ELISA Evaluation

4.2.1 Sensitivity

4.2.2 Specificity

4.2.3 Accuracy

4.2.4 Limit of Detection (LOD)

4.3 Measurable Units in ELISA

References

5 Advantages, Disadvantages and Modifications of Conventional ELISA

Abstract

5.1 Significance of Conventional ELISA

5.2 Shortages of Conventional ELISA

5.3 Materials of Choice for Fabrication of ELISA Well Plates

5.4 Different Types of ELISA Well Plates

5.5 Modified ELISA Platforms

5.5.1 ELISA on Coated Platforms

5.5.2 ELISpot

5.5.3 Plasmonic ELISA

5.5.4 Sphere-/Bead-Based ELISA

5.5.5 Paper-Based ELISA

5.5.6 Fiber-Based ELISA

5.5.7 ELISA in Micro-Devices

5.5.8 Other Strategies

5.5.8.1 Smart Devices in ELISA

5.5.8.2 Digital ELISA

5.5.8.3 Aptamers as Antibody Substitutes in ELISA

5.5.8.4 Multiple and Portable ELISA

References