Immunoassays

Development, Applications and Future Trends

Richard O'Kennedy, Caroline Murphy, Richard O'Kennedy, Caroline Murphy

• Publisher: Jenny Stanford Publishing
• Year: 2017
• Print ISBN: 9789814669979
• eBook ISBN: 9781351797344
• Topic: Medicine
• Subtopic: Biotechnology in Medicine

The concept behind this book is to provide a detailed and practical overview of the development and use of immunoassays in many different areas. Immunoassays are analytical tests that utilise antibodies to measure the amount, activity or identity of an analyte. This book 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. It is comprised of 13 chapters written by highly experienced researchers in the fields of antibody-based research, immunoassay development, assay validation, diagnostics and microfluidics.

Beginning with a comprehensive survey of antibodies, immunoassay formats and signalling systems, the book elucidates key topics related to the development of an ideal antibody-based sensor, focuses on the important topic of surface modification, explores key parameters in the immobilisation of antibodies onto solid surfaces, discusses the move to 'lab-on-a-chip'-based devices and investigates the key parameters necessary for their development. Three of the chapters 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, including next-generation immunoassays, electrochemical-immunoassays and 'lab-on-a-chip'-based systems, is also discussed. The book also covers the use of optical detection systems (with a focus on surface plasmon resonance) in immunoassays, provides a compilation of important, routinely used immunoassay protocols and addresses problems that may be encountered during assay development.

Chapter 1

An Overview of Immunoassays

Caroline Murphy, Sarah Gilgunn, and Richard O’Kennedy

School of Biotechnology and Biomedical Diagnostics Institute, Dublin City University, Collins Avenue, Dublin 9, Ireland

[email protected]

The aim of this chapter is to provide a detailed and practical introduction to immunoassays and set the overall context for the other detailed chapters on specific key elements of immunoassays. The production of antibodies, various antibody structures and their fundamental role in immunoassays is outlined. Comprehensive guides to different immunoassay formats ranging from direct to competitive are provided, and subsequently, important signalling systems, including colourimetric and fluorescence-based approaches, are examined. Finally, electrical, mechanical and optical signal transduction mechanisms that are used in the next generation of immunoassays are described.

1.1 Introduction to Antibodies and Immunoassays

Immunoassays are biochemical tests that utilise immunoglobulins (Ig) (antibodies) as high sensitivity binders to detect the presence of molecules that are present at low concentrations. Antibodies are an integral part of the humoral immune system and are nature’s major recognition devices. They are found on the surface of B-cells (B-lymphocytes) and recognise the presence of foreign antigens. The B-cell can detect and signal the presence of an extensive range of antigens, consisting of molecules ranging from prions to viruses, drugs, toxins and aberrant biomolecules that invade our bodies. Immunoglobulin proteins exist in many different formats, the five main isotypes of immunoglobulin are IgG (shown in Fig. 1.1), IgM, IgD, IgA and IgE.

Immunoassays: Development, Applications and Future Trends

Edited by Richard O’Kennedy and Caroline Murphy

Copyright © 2017 Pan Stanford Publishing Pte. Ltd.

ISBN 978-981-4669-97-9 (Hardcover), 978-1-315-20654-7 (eBook)

www.panstanford.com

Figure 1.1 Structure of immunoglobulin G (IgG). IgG is made up of two heavy chains (composed of constant domains C_H1, C_H2 and C_H3 and variable heavy domain, V_H), and two light chains (composed of the constant light domain, C_L and the variable light domain, V_L), connected by disulphide bonds. The antigen-binding region is composed of a variable heavy chain and a variable light chain, which recognises the epitope (specific area where binding occurs) of the antigen.

Antibodies are at the forefront of targeted therapeutics and diagnostics due to their natural high affinities and excellent half-lives. They can be readily manipulated using standard molecular biological techniques into customised antibodies that are tailored to perform efficiently in their chosen end-point application. The biopharmaceutical industry has heavily invested in antibody-based diagnostics and therapeutics, and the latter currently represents the largest and fastest growing class of biopharmaceuticals [1, 2].

Antibodies are generally represented in three forms: (i) poly-clonal (produced from a mixture of various B-cell clones), (ii) monoclonal (secreted from a single clone of B-cells) and (iii) recombinant antibodies (the product of the genetic manipulation of antibody genes) [3, 4].

1.1.1 Polyclonal Antibody Production

Polyclonal antibody production involves the immunisation of animals with an antigen and adjuvant (immune system stimulating material) mixture, resulting in the activation of multiple B-cells targeting different antigen epitopes. This produces a vast number of antibodies with different specificities and epitope affinities [3]. These polyclonal antibodies are purified from the serum of immunised animals. Polyclonal antisera can be obtained relatively quickly (6–12 weeks for highly immunogenic antigens) but the time may be antigen-dependent. Methods of purification are generally associated with the antibody type and the intended application(s) for the antibody. These are listed in Table 1.1 [5] and are discussed in detail in Chapter 12.

Several key steps need to be considered for the production of polyclonal antibodies, including (i) preparation/availability of antigen, (ii) choice of host species, (iii) injection regime, (iv) monitoring antibody response to immunogen and (v) collection/purification of antibodies. The choice of host is of particular importance when developing polyclonal antibodies against human derived targets, as a large number of proteins are highly conserved throughout mammalian evolution, and, are, therefore, common to many mammalian species. Hence, immunisation of such proteins into rabbits and mice, may generate a limited immune response [6]. The use of a species more phylogenetically distant from humans such as chickens (that diverged from mammalian genomes some 310 million years ago [7]) is an ideal alternative for immunisation and selection of antibodies against highly conserved human proteins [6, 8].

Table 1.1 Polyclonal antibody purifiation methods [5]

Purification type	Methodologies involved in the purification
Crude	Precipitation of a subset of total serum proteins that includes immunoglobulins.
General	Affinity purification of certain antibody classes (e.g. IgG).
Specific	Affinity purification of only those antibodies in a sample that bind to a particular antigen molecule.

1.1.2 Production of Monoclonal Antibodies

The development of ‘hybridoma technology’ won Georges Köhler and César Milstein the Nobel Prize in Physiology or Medicine in 1984. They created an immortal cell line capable of producing an endless supply of identical antibodies with known specificity called ‘monoclonal antibodies’ signifying the fact that they were derived from a single hybrid cell [9]. The production of monoclonal antibodies is shown in Fig. 1.2 [3].

Figure 1.2 Production of monoclonal antibodies by hybridoma technology. B-cells from an immunised mouse are isolated and fused with a myeloma cell line creating hybridomas (generally mediated by the addition of polyethylene glycol (PEG)). Cloning and selection of a specific hybridoma is carried out by ‘limiting dilution’. The clones are screened for reactivity and specific antibody-producing clones are further characterised and expanded for generation of the desired antibody.

1.1.3 Recombinant Antibody Fragments

Recombinant antibodies are highly attractive for customised antibody development as genetic manipulations can be easily employed to precisely tailor their specificity and biophysical properties [10–14]. The ability to readily generate bulk quantities of recombinant proteins, with low production costs in Escherichia coli, has fuelled the emergence of an assortment of distinct antibody constructs that can be employed in a wide variety of applications. Some common recombinant antibody constructs are shown in Fig. 1.3.

The smallest antibody fragment that retains the full monovalent antigen binding capabilities of a human IgG is the variable fragment (Fv) and it is comprised of the variable heavy (V_H) and variable light (V_L) domains held together by non-covalent interactions [12, 13]. The single chain fragment variable (scFv) is more stable than the Fv (which is more prone to aggregation than the scFv) and typically, a flexible 15 residue glycine/serine ((Gly₄Ser)₃) linker is incorporated to link the chains together and aid antibody folding and stability during bacterial expression [14].

Figure 1.3 Antibody fragment constructs. The mammalian IgG construct (150 kDa) consists of two heavy and two light polypeptide chains linked together by disulphide bonds. The heavy chain consists of variable heavy (V_H) and three constant (C_H1, C_H2, and C_H3) regions. The scFv consists of the V_H and V_L chains joined together by a flexible linker. A single chain antibody (scAb) fragment consists of a scFv with an additional constant chain (either heavy or light). The Fab (antigen-binding fragment) contains the full light chain with V_H and C_H1. F(ab’)₂ is comprised of two Fab fragments held together by disulphide bonds.

The antigen-binding fragment (Fab) is a larger, more stable fragment of approximately 50 kDa. The Fab fragment consists of the variable heavy, variable light, constant heavy and constant light chains (V_H & C_H1 and V_L & C_L). An interchain disulphide bond exists between the C_H1 and C_L (Fig. 1.3) [15].

There are various advantages and disadvantages associated with the different types of recombinant antibody structures that can be engineered. It should also be noted that antibody fragments can be relatively easily reformatted for improved characteristics such as expression [16], reduction in non-specific binding [17] and enhanced specificity [18]. The selection of superlative antigen-specific recombinant fragments can be achieved using a process known as phage display technology.

1.1.3.1 Production of recombinant antibodies by phage display technology

When George P. Smith first demonstrated in 1985, that the link between phenotype and genotype could be established in filamentous bacteriophage, the advent of phage display technology emerged [19]. Over the last few decades an increased understanding of antibody structure and function has allowed phage display to become one of the most powerful tools in the controlled selection ...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Contents
• Preface
• Glossary
• 1 An Overview of Immunoassays
• 2 Recombinant Antibodies for Diagnostic Applications: Design Considerations and Structural Correlates
• 3 Surfaces and Immobilisation Strategies for Use in Immunoassay Development
• 4 Immunoassay Validation
• 5 Lab-on-a-Chip Immunoassay Systems
• 6 Clinical Applications of Immunoassays
• 7 Immunoassay-Based Detection of Infectious and Parasitic Diseases
• 8 Detection of Food, Agricultural and Aquatic Contaminants
• 9 Next-Generation Immunoassays
• 10 Recent Trends and the Future of Electrochemical Immunoassay Systems
• 11 Optical Signal Transduction with an Emphasis on the Application of Surface Plasmon Resonance (SPR) in Antibody Characterisation
• 12 Protocols for Key Steps in the Development of an Immunoassay
• Index