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Immunoassays
Pan Stanford Series on Renewable Energy — Volume 2

Immunoassays
Development, Applications and Future Trends

edited by
editors
Preben Maegaard Richard 0’Kennedy
Anna Krenz
Wolfgang Palz
Caroline Murphy

The Rise of Modern Wind Energy

Wind Power
for the World
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Published by
Pan Stanford Publishing Pte. Ltd.
Penthouse Level, Suntec Tower 3
8 Temasek Boulevard
Singapore 038988

Email: [email protected]
Web: www.panstanford.com

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Immunoassays: Development, Applications and Future Trends

Copyright 
c 2017 Pan Stanford Publishing Pte. Ltd.

All rights reserved. This book, or parts thereof, may not be reproduced in any
form or by any means, electronic or mechanical, including photocopying,
recording or any information storage and retrieval system now known or to
be invented, without written permission from the publisher.

For photocopying of material in this volume, please pay a copying

fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive,
Danvers, MA 01923, USA. In this case permission to photocopy is not
required from the publisher.

ISBN 978-981-4669-97-9 (Hardcover)

ISBN 978-1-315-20654-7 (eBook)

Printed in the USA

April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Contents

Preface xv
Glossary xvii

1 An Overview of Immunoassays 1
Caroline Murphy, Sarah Gilgunn, and Richard O’Kennedy
1.1 Introduction to Antibodies and Immunoassays 1
1.1.1 Polyclonal Antibody Production 3
1.1.2 Production of Monoclonal Antibodies 4
1.1.3 Recombinant Antibody Fragments 5
1.1.3.1 Production of recombinant antibodies
by phage display technology 6
1.2 Types of Immunoassays 7
1.3 Choosing a Suitable Immmunoassay Technique 9
1.3.1 Detection of Target Analyte: An Immunometric
Approach 9
1.3.1.1 Direct immunoassay 9
1.3.1.2 Indirect immunoassay 10
1.3.1.3 Sandwich immunoassay 11
1.3.2 Detection of Small Molecules Using a
Competitive Approach 12
1.4 Signal Generation in Immunoassays 14
1.4.1 Signal Generation 14
1.4.1.1 Colourimetry as a method of
immuno-detection 14
1.4.1.2 Luminescence as a method of
immuno-detection 18
1.4.2 Nanomaterials, for Signal Generation and
Immobilisation 21
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

vi Contents

1.5 Next Generation Signal Transduction Systems 23

1.5.1 Electrochemical Transducers for Immunoassay
Applications 24
1.5.2 Nanomechanical Sensors/Piezoelectric Mass
Sensors 24
1.5.3 Optical Signal Transduction 27
1.6 Conclusions and Key Issues 27

2 Recombinant Antibodies for Diagnostic Applications: Design

Considerations and Structural Correlates 37
Stephen Hearty and Richard O’Kennedy
2.1 Diagnostic Antibodies: Co-opting Natural Immunity 38
2.2 Harnessing Natural Diversity 38
2.3 Affinity 41
2.4 Exploiting Synthetic Diversity in vitro 43
2.4.1 Introducing Diversity 44
2.4.2 Introducing Diversity: Affinity Maturation 46
2.5 Paradigms Challenged 47
2.6 Single-Domain Antibodies 49
2.7 Conclusion 53

3 Surfaces and Immobilisation Strategies for Use in

Immunoassay Development 59
Chandra K. Dixit and Paul Conroy
3.1 Introduction 60
3.2 Tailoring Immunoassay Surfaces 63
3.2.1 Surface Designing/Modulation and
Customisation for Antibody Immobilisation
and Immunoassay Development 64
3.2.1.1 Precursor modulation and
post-manufacture treatments for
controlling the surface properties 64
3.2.1.2 Grafting functional groups by physical
and chemical methods 69
3.3 Antibody Immobilisation 71
3.4 Antibody Conjugation to Quantum Dots 75
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Contents vii

3.5 Aspects and Case Studies of Antibody Immobilisation 76

3.6 Conclusions and Future Perspectives 80

4 Immunoassay Validation 89
Paul Leonard
4.1 Why Is Validation Necessary? 90
4.2 Assay Validation Overview 90
4.2.1 Planning ‘It Takes Time to Save Time’ 91
4.3 Assay Development 92
4.3.1 Immunogen for Antibody Production and
Screening 95
4.3.2 Antibody Production 96
4.3.3 Assay Format 96
4.4 Pre-validation 97
4.4.1 Select Assay Reagents 97
4.4.2 Evaluate Assay Conditions 97
4.4.3 Define Calibration Model 98
4.4.4 Evaluate Selectivity and Specificity 99
4.4.5 Preliminary Estimates for LLOQ and ULOQ 100
4.4.6 Preliminary Estimates of Precision and
Accuracy 101
4.4.7 Estimation of the Assay Limit of Detection
(LOD) 104
4.4.8 Reference Method 107
4.4.9 Validation Samples and Method Definition 108
4.4.10 Evaluate Extraction Efficiency/Effects 108
4.5 Pre-study Validation 110
4.5.1 Stability 110
4.5.2 Evaluate Dilutional Linearity 111
4.5.3 Evaluate Assay Robustness and Ruggedness 112
4.5.4 Assay Validation Report and Documentation 112
4.6 Summary 113

5 Lab-on-a-Chip Immunoassay Systems 117

Barry Byrne and Louise M. Barrett
5.1 Introduction 118
5.2 Substrates Used for Microfluidic Chip Design 119
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

viii Contents

5.3 Microfluidic Channel Design and Fabrication 122

5.4 Analyte Delivery, Mixing and Washing 125
5.5 Antibody Integration 128
5.6 Lab-on-a-Chip–Based Immunodetection: Applications 132
5.6.1 Bacterial Pathogens and Toxins 132
5.6.2 Marine and Fungal Toxins 140
5.7 Disease Markers and Their Detection Using
Microfluidics-Based Devices 145
5.8 Conclusions 148

6 Clinical Applications of Immunoassays 161

Karen Lawler and Gregor Kijanka
6.1 Clinical Specimen Analysis 162
6.2 Cardiovascular Disease 165
6.2.1 Ischemic Heart Disease and Cardiac Enzymes 165
6.2.2 Heart Failure and Brain Natriuretic Peptide 166
6.2.3 Serum Lipids and Lipoproteins 167
6.3 Respiratory Disease 168
6.3.1 Bacterial Respiratory Infections 168
6.3.1.1 Streptococcus pneumonia 168
6.3.1.2 Legionella species 169
6.3.1.3 Mycoplasma pneumonia 169
6.3.1.4 Haemophilus influenzae 170
6.3.1.5 Chlamydia pneumonia 170
6.3.2 Fungal Respiratory Infections 170
6.3.2.1 Aspergillus 170
6.3.3 Viral Respiratory Infections 171
6.3.3.1 Influenza A and B 171
6.3.4 Cystic Fibrosis 171
6.3.5 Pulmonary Embolism and D-Dimers 172
6.4 Endocrinology 173
6.4.1 Type 1 Diabetes Mellitus 173
6.4.2 Thyroid Disease 173
6.4.3 Adrenal Hormone and Cushing’s Syndrome 174
6.5 Rheumatology 175
6.5.1 Rheumatoid Arthritis 175
6.5.2 Systemic Lupus Erythematosus 176
6.5.3 Systemic Sclerosis 177
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Contents ix

6.5.4 Sjögren’s Syndrome 177

6.5.5 Vasculitis 178
6.5.6 Dermatomyositis and Polymyositis 178
6.6 Oncology 179
6.6.1 Alpha-Fetoprotein 179
6.6.2 Prostate-Specific Antigen 180
6.6.3 Carbohydrate Antigens 181
6.6.3.1 CA 125 181
6.6.3.2 CA 15-3 and CA 27-29 181
6.6.3.3 CA 19-9 181
6.6.4 Carcinoembryonic Antigen 181
6.6.5 Beta-Human Chorionic Gonadotrophin 182
6.6.6 Antibody-Associated Paraneoplastic Disease 182
6.7 Conclusion 183

7 Immunoassay-Based Detection of Infectious and Parasitic

Diseases 191
Paul J. Conroy
7.1 Introduction 192
7.2 Current Technologies for the Detection of Infectious
Diseases in Humans 193
7.2.1 Appropriate Technologies 196
7.2.2 Human Immunodeficiency Virus (HIV) 197
7.2.3 Tuberculosis 198
7.2.4 Malaria 199
7.2.5 Rapidly Spreading Infectious Agents 200
7.3 Challenges in Detecting Infectious and Parasitic
Diseases 202
7.3.1 Current Research into New Platform
Development 202
7.3.2 Current Antibodies for Infectious Disease
Agents 205
7.4 Future Trends 207
7.4.1 Rapid Assay Development: Antibodies 207
7.4.2 Rapid Assay Development: Microfluidics and
Nanotechnology and Their Relevance to
Infectious Disease Detection 209
7.5 Conclusion 212
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

x Contents

8 Detection of Food, Agricultural and Aquatic Contaminants 221

Marie Le Berre, Caroline Viguier, Caroline Murphy, and
Niamh Gilmartin
8.1 Immunoassays Used in the Detection of Contaminants
in Food 222
8.1.1 Foodborne Pathogens 222
8.1.2 Foodborne Toxins 227
8.2 Immunoassays in the Agricultural Environment 228
8.2.1 Immunoassays in Animal Feedstuffs 228
8.2.2 Immunoassays for Agricultural Veterinary
Infections 232
8.2.3 Immunoassays Used to Assist in the
Management of Reproduction in Animals 233
8.3 Immunoassays in Aquatic Environments 235
8.3.1 Detection of Marine Toxins 235
8.3.2 Detection of Freshwater Algal Toxins 239
8.4 Conclusion 242

9 Next-Generation Immunoassays 255

Valerie Fitzgerald and Paul Leonard
9.1 History of the Immunoassay 255
9.2 Evolution of the Immunoassay 256
9.3 Improvements in Antibody Development 258
9.3.1 Antibody Engineering 259
9.3.1.1 Phage display 260
9.3.1.2 Cell display methods 261
9.3.1.3 In vitro display technologies 263
9.3.2 Mutagenesis Strategies 264
9.4 Improvements in Platform and Detection Methods 266
9.4.1 Nanomaterials 266
9.4.2 Nucleic Acid–Based Signal Amplification 269
9.4.2.1 Immuno-PCR 269
9.4.2.2 Hybridisation chain reaction 271
9.4.2.3 Loop-mediated isothermal
amplification 272
9.4.3 Optical Detection Methods 273
9.5 Current Immunoassay Trends 275
9.5.1 Point-of-Care (POC) Diagnostics 275
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Contents xi

9.5.2 Microfluidics 276

9.6 Commercially Available Novel Immunoassays 278
9.7 Conclusion 282

10 Recent Trends and the Future of Electrochemical

Immunoassay Systems 291
Frank Davis and Seamus Higson
10.1 Introduction to Electrochemical Immunoassays 292
10.2 Electrochemical Immunoassays 294
10.2.1 Principle of Electrochemical Immunoassay 295
10.2.2 Labelled Immunoassay Methods 296
10.2.3 Label-Free Methods 298
10.3 Use of Nanomaterials 301
10.3.1 Metal Nanoparticles 301
10.3.2 Carbon Nanotubes 303
10.3.3 Other Nanomaterials 306
10.4 Microarrays and Nanoarrays 307
10.5 Multiple Target Detection 310
10.6 Imaging of Arrays and Scanning Electrochemical
Microscopy (SECM) 312
10.7 Conclusions and Future Prospects 314

11 Optical Signal Transduction with an Emphasis on

the Application of Surface Plasmon Resonance (SPR)
in Antibody Characterisation 327
Caroline Murphy, Aoife Crawley, Hannah Byrne,
Kara Moran, Jenny Fitzgerald, and Richard O’Kennedy
11.1 The Fundamentals of Optical Signal Transduction 328
11.2 Description and Evaluation of the Use of Surface
Plasmon Resonance in Immunoassays 330
11.2.1 Optical Platforms Used in SPR 333
11.3 Fundamental Steps in SPR-Based Analysis 335
11.4 Surfaces Used in SPR Immunoassay Development 336
11.4.1 Dextran-Coated Sensor Chips: The
Carboxymethylated Series 337
11.4.2 Sensor Chip with Pre-immobilised
Surfaces: Capture Applications 337
11.4.3 Sensor Chips for Specialised Applications 340
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

xii Contents

11.4.4 Functionalisation of SPR Surfaces 341

11.4.5 The Use of Reference/Control Surfaces in
SPR Immunoassays 343
11.5 Determination of Kinetic Parameters between
Binding Partners Using SPR 344
11.6 Immobilisation of Ligands onto Sensor Surfaces 347
11.6.1 Electrostatic Pre-concentration of the
Protein onto the Sensor Chip 348
11.7 Regeneration of Surfaces 348
11.8 Advances in SPR Immunoassays 349
11.9 Conclusions 353

12 Protocols for Key Steps in the Development of an

Immunoassay 361
Caroline Murphy and Richard O’Kennedy
12.1 Introduction: Immunoassay Protocols 361
12.2 Preparation of Sample Analyte from Complex
Matrices 363
12.3 Purification of Antibodies 364
12.3.1 Purification of Antibodies by
Chromatography 366
12.3.2 Antibody Storage Considerations 371
12.3.3 Antibody Conjugation 372
12.3.3.1 Conjugation of quantum dots to
antibodies 376
12.3.4 Preparation of Biotinylated Antibodies 379
12.4 Protein Aggregation 382
12.4.1 Prevention of Protein Aggregation 382
12.5 Immunoassay Optimisation 384
12.5.1 Immunoassay Buffers 384
12.5.2 Decreasing Background Signal 384
12.5.3 Coating Buffer 385
12.5.4 Inclusion of Wash Steps 386
12.5.5 Blocking of Non-specific Reaction Sites 386
12.5.6 Checkerboard Titration for Optimisation of
ELISA Components 388
12.5.7 Introduction of Appropriate Positive and
Negative Controls into the Assay Protocol 390
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Contents xiii

12.5.7.1 Use of a spiked control 391

12.5.7.2 Standard curve 391
12.5.8 Immobilisation Strategies for Biomolecules
onto Solid Surfaces 391
12.6 What Type of Immunoassay to Choose? 395
12.6.1 Enzyme-Linked Immunosorbent Assay
(ELISA) 395
12.6.1.1 Binding of capture protein/
antibody to the surface 395
12.6.2 Commonly Used ELISA Protocols 396
12.6.3 Fluorescence-Linked Immunosorbent
Sandwich Assay (FLISA) 405
12.6.4 Time-Resolved Fluorescence Immunoassay 406
12.6.5 Fluorescence Polarisation Immunoassay
(FPIA) 408
12.6.6 Fluorescence Resonance Energy Transfer
(FRET) Immunoassay 410
12.7 Surface Plasmon Resonance Protocols 411
12.8 Trouble Shooting 418
12.8.1 Binding of Protein to Surfaces 418
12.8.2 ELISA Trouble Shooting 419
12.9 Summary 420

Index 425
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Preface

Immunoassays are analytical tests that utilise antibodies to measure

the amount, activity or identity of an analyte. The concept behind
this book is to provide a detailed and practical overview of the
development and use of immunoassays in many different areas.
It is designed to provide a critical and helpful insight into the
subject and to give the user practical information that may be of
assistance in assay format selection, antibody generation/selection
and choice of appropriate detection strategies. This book comprises
12 chapters written by highly experienced researchers in the
fields of antibody-based research, immunoassay development, assay
validation, diagnostics and microfluidics.
Chapter 1 is a comprehensive survey of antibodies, immunoassay
formats and signalling systems. Chapter 2 elucidates key topics
related to the development of an antibody-based sensor. Chapter 3
focuses on the important topic of surface modification and explores
key parameters in the immobilisation of antibodies onto solid
surfaces. Chapter 4 provides a detailed account of what is involved
in immunoassay validation. Chapter 5 discusses the move to
‘lab-on-a-chip’-based devices and investigates the key parameters
necessary for their development. Chapters 6, 7 and 8 are dedicated
to the areas of clinical diagnostics, infectious disease monitoring
and food security, where immunoassay-based applications have
become highly valuable tools. The future of immunoassays and
electrochemical-based detection systems are discussed in Chapters
9 and 10. Chapter 11 concentrates on the use of optical detection
systems (with a focus on surface plasmon resonance) in immunoas-
says. Finally, Chapter 12 is a compilation of important, routinely
used, immunoassay protocols and addresses problems that may be
encountered during assay development.
April 21, 2017 8:57 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

xvi Preface

We would like to sincerely thank all the authors involved, the

Applied Biochemistry Group for their help reviewing the chapters
and Garvan Doherty, from the Biomedical Diagnostics Institute, for
his help in developing the images used in the book.
The support of the publishers and their staff in relation to the
initiation, evolution and execution of the text and their patience is
gratefully acknowledged.
The financial support of Dublin City University, the Biomedical
Diagnostics Institute and Science Foundation Ireland through Grant
No. 10/CE/B1821 and 14/IA/2646 was vital for the completion of
this work.
Finally, Caroline would like to thank her family, Mary, Claire,
Aisling, Rosemary, Pauric, Dicky, her husband George and her
daughter Fia for all their support, and Richard would like to thank
his family, Kristina, Molly and Jack, for their support and enduring
patience during the preparation of this book.

Caroline Murphy
Richard O’Kennedy
January 2017
May 13, 2017 15:9 PSP Book - 9in x 6in 00-O’Kennedy-Prelims

Glossary

If words are not explained at first sight in the book, they are
highlighted in bold and are explained in the glossary.

AC impedance Alternating current impedance is a measure of the

opposition to the flow in an AC electrical current.
Accuracy Accuracy is a measure of the closeness of agreement of
a test result obtained by an analytical method to its theoretical
true value, or the accepted reference/standard value.
Affinity The affinity of an antibody refers to its ability to bind to the
target antigen. It is a function of how well the antigen fits the
binding site of the antibody.
Affinity ceiling Antibody recognition is thought to have an
upper boundary for optimal affinity under natural biological
conditions. This is called an affinity ceiling. Natural equilibration
dissociation constant (K D ) is considered to be in the order of
10−8 to 10−10 .
Amino acids Amino acids are organic compounds that are com-
posed of an amine (–NH2 ) group and carboxyl (–COOH) group.
They are the building blocks of proteins. In proteins, specific
amino acids are linked together as if in a chain, and when folded,
the tertiary structure makes up a protein.
Amperometric Amperometric analytical signalling systems operate
when a current is created as a result of redox reactions at a
sensor surface. They rely on either the release or consumption
of electrons during these reactions.
Analyte Any chemical or substance under analytical scrutiny.
Anthelmintics Anthelmintics are a class of drugs used to treat
infections with parasitic worms (helminths) from the body.
Tapeworms, roundworms and flukes are classified as helminths.
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