IMMUNOASSAY FUNDAMENTALS

P A R T 1

3© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00001-4

The Immunoassay Handbook is a comprehensive source of information about immunoassays and their applications. This chapter explains how the book is structured to help you find the information you need.

Using the IndexIn the index, if a page reference is in bold type, the topic is usually explained in more detail.

Book StructureThe book content is separated into parts, each one build-ing on the previous body of information.

PART 1 – IMMUNOASSAY FUNDAMENTALS

1.2 Immunoassay for beginners is a good place to start for newcomers, explaining the basic principles of immunoassay.

1.3 Immunoassay performance measures explains the terminology and methodology that define the performance of an immunoassay.

PART 2 – IMMUNOASSAY CONFIGURATIONS

2.1 Principles of competitive and immunometric assays (including ELISA). Nearly all immunoassays are competi-tive or immunometric in configuration. They are explained in the same chapter because they share some common characteristics. ELISA (Enzyme-Linked Immunosorbent Assay) is a subgroup of immunometric assays although the term is sometimes used loosely (but incorrectly) to describe any immunoassay. Immunometric assays are commonly known as sandwich assays because the analyte becomes sandwiched between the two antibodies (the capture and labeled antibodies).

2.2 Non-competitive immunoassays for small mole-cules. Usually the competitive assay configuration is used for small molecules, such as steroid hormones and many drugs, because of the technical difficulties involved in cre-ating an antibody sandwich with the relatively small ana-lyte between them. But immunometric assays have significant performance advantages. This chapter explains how immunometric assay performance can be achieved with low-molecular-weight analytes by creating a pseudo-immunometric assay format.

2.3 Homogeneous immunoassays. Conventional immu-noassays are heterogeneous, relying on separation of mate-rial bound to antibody from the unbound material, so that

only the bound fraction results in a measurable signal from the label. Homogeneous immunoassays are designed to discriminate bound from unbound label without requiring a separation, by a range of ingenious approaches. This simplifies assay automation. They can be competitive or immunometric. This is an innovative area, receiving con-siderable attention from researchers.

2.4 Lateral flow immunoassay systems. This type of immunoassay is widely used and deserves a special chapter. A well-known example is the home pregnancy test. The format is used in point-of-care, forensic, environmental, and food applications. A key characteristic is accomplish-ment of the mechanical steps, including separation, on an inert test strip through capillary action without interven-tion by the user. This is a field that is the subject of inten-sive development activity.

2.5 Ambient analyte immunoassay. Professor Roger Ekins applied immunoassay theory to achieve something that intuitively sounds impossible: a quantitative assay that is independent of the sample volume. Small, immobi-lized microdots of antibody sample the analyte in solution, rather like a thermometer measuring the surrounding temperature without affecting it.

2.6 Free analyte immunoassay. This is a special family of assay formats, designed to measure free analyte in blood samples, i.e., the fraction that is not bound to carrier pro-tein. This is important if the free analyte is the metaboli-cally active fraction. Free thyroxine is the best known example, with a free fraction of just 0.02% of the total.

2.7 Qualitative immunoassays—features and design. As immunoassays have evolved to meet specific clinical needs, qualitative tests that give a yes/no answer have become more common. Home pregnancy tests are qualita-tive as are some blood-screening tests, which sometimes also have a gray zone between the positive and negative classification areas. Although qualitative immunoassays are simple for the user, they have unique design requirements, because the test provides clinical interpretation, rather than simply the analyte concentration.

2.8 Detection of antibodies relevant to infectious dis-ease. Conventional immunoassays utilize antibodies to “fish” for complementary antigens: the target analytes. The same principles apply when antibodies are the targets, and antigens are used as the “bait,” but in practice anti-body assays have unique challenges. In a conventional immunoassay, the developer can select a clone or pool of antibodies to engineer the desired assay performance char-acteristics. But in an antibody assay, the antibody popula-tions may be highly variable between samples.

2.9 Microsphere-based multiplex immunoassays: devel-opment and applications using Luminex® xMAP® tech-nology. Luminex pioneered the use of antibody-coated microspheres and cell sorting, and this is now an important immunoassay class, with many applications.

How to Use This BookDavid Wild (david@davidwild.net)

C H A P T E R

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4 The Immunoassay Handbook

2.10 Lab-on-a-chip, micro- and nanoscale immunoassays. This chapter explains how the relation-ships between physical forces change when assays are min-iaturized, e.g., the greater impact of surface tension and the reduced influence of gravity. The sample-loading part of the device, which needs to be on a human scale, may dwarf the assay module in size. Every aspect of an immunoassay needs to be re-engineered to achieve miniaturization. This is a very interesting area of science and engineering.

2.11 Immunological biosensors. Biosensors measure the binding of antibody to antigen—the crucial event in an immunoassay—directly, creating or attenuating a signal. Eventually this method could allow substances to be mea-sured in situ, without a sample being required.

2.12 Surface plasmon resonance in binding site, kinetic, and concentration analyses. Surface plasmon resonance is a biosensor technique that has been refined to provide an insight into the binding kinetics of an antibody–antigen pair, which is at the heart of every immunoassay. In this chapter, the importance of the association and dissociation constants, and how their effects may be visualized, are explained.

2.13 Measurement of single protein molecules using Dig-ital ELISA. This is an exciting new single molecule assay for-mat that has achieved remarkable results, measuring extremely low concentrations of polypeptides with precision.

PART 3 – IMMUNOASSAY COMPONENTSThere are many permutations of immunoassay methods, but they possess common elements. Once the basic com-ponents are understood, it is simpler to understand any novel immunoassay system.

3.1 Antibodies. Most immunoassays utilize antibodies as the most critical reagent (some immunoassays test for the presence of specific antibodies in samples using antigen instead). This chapter explains how antibodies are har-nessed as exquisitely selective reagents and the recombi-nant techniques available. Some of the techniques described are also contributing to a revolution in the phar-maceutical industry, through the use of humanized mono-clonal antibodies as therapeutic drugs.

3.2 Signal generation and detection systems. In order to quantify the binding of the antibody to the target ana-lyte, a signal is needed. This chapter explains the most common signal generation systems, with their advantages and limitations.

3.3 Solid-phase and other separation systems. Separa-tion removes surplus, unbound signal generation material so that only the bound signal is measured. It has a direct impact on immunoassay sensitivity. This chapter explains the consequences of using solid phases to anchor the cap-ture antibody, which simplifies separation but can have implications for antibody behavior and assay kinetics.

3.4 Conjugation methods. The antibody–analyte–anti-body complex formed in the presence of analyte in an immu-nometric assay is useless without a signal-generating entity to quantify it. In most immunoassays this involves conjuga-tion of one of the antibodies to a molecular label, which can participate in a signal generation system. In an ELISA, the label is an enzyme. This is one of several situations in immu-noassay where an antigen or antibody needs to be chemically

conjugated with another molecule. It is a specialized area of immunoassay that is explained in this chapter.

3.5 Standardization and calibration. Immunoassay is an indirect measurement system that requires calibration to provide meaningful estimates of concentration. In some cases, the calibration is referenced to a standard with a meaningful unit of measurement, such as moles or grams, which can be verified, but in others, standardization is against arbitrary units. This chapter explains how immu-noassays are standardized and calibrated.

3.6 Calibration curve fitting. This chapter explains the models and algorithms used to computerize the process of reading unknown sample concentrations from standards or calibrators.

PART 4 – RELATED TECHNIQUES4.1 The foundations of immunochemistry. This chapter

looks back to the period before the invention of immuno-assay (as defined in this book) to other antibody-based methods and places them in a historical context. Mile-stones highlighted in this chapter date back to 1895. Figures from oft-quoted landmark papers have been located in libraries by the author and digitally restored.

4.2 Immunohistochemistry and immunocytochemistry. Immunohistochemistry and immunocytochemistry are so closely related to immunoassay that they are included in this book. In the field of proteomics, these techniques are used to study proteins of scientific interest in cells, dis-cover where they are located, compare diseased and nor-mal cells for clues about causation and possible treatments, and to evaluate one treatment against another at the cellular level.

PART 5 – IMMUNOASSAY DEVELOPMENT5.1 Practical guide to ELISA development. “Home-

brew” immunoassays can be used to screen thousands of samples cheaply and simply, one of the great attractions of immunoassay. This is a hands-on, practical guide to ELISA development, intended for researchers and those with lim-ited resources.

5.2 Method evaluation—a practical guide. This is a guide to initial assay evaluation for users of commercial products.

5.3 Interferences in immunoassay. There are many potential causes of interference in immunoassays, deriving from samples and other sources. This is a comprehensive guide to the types of interference, and how to detect and prevent them.

5.4 Immunoassay development in the in vitro diagnos-tic industry. This is a description of commercial immuno-assay development written by industry insiders.

PART 6 – IMMUNOASSAY IMPLEMENTATION

6.1 Sample collection, including participant prepara-tion and sample handling. This chapter provides compre-hensive advice about subject preparation, sample collection, and other related topics. It covers blood and other types of sample, with extended coverage of saliva collection.

5CHAPTER 1.1 How to Use This Book

6.2 Laboratory quality assurance. This is a guide to cre-ating a secure quality system in immunodiagnostic laboratories.

6.3 Point-of-care testing. This chapter covers the man-agement of point-of-care tests in clinical practice and con-siderations for decision makers about when, and when not, to apply them.

6.4 Choosing an automated immunoassay system. This is a guide to laboratory managers on the criteria for select-ing an automatic analyzer.

6.5 Immunoassay troubleshooting guide. A compre-hensive and systematic guide to identifying the root causes of problems with immunoassays.

PART 7 – IMMUNOASSAY PRODUCT TECHNOLOGY

7.1 Introduction to immunoassay product technology in clinical diagnostic testing. This is an introduction to the technologies behind the contemporary immunoassay sys-tems described in Part 7 of the book, with some back-ground on groundbreaking products of the past.

7.2 Market trends. This chapter describes the clinical diagnostics immunoassay market, its growth and market share, and likely trends for the future.

7.3 Lateral flow and consumer diagnostics. This chap-ter describes the pregnancy and ovulation test range origi-nally developed by Unipath and now marketed by SPD Swiss Precision Diagnostics (SPD).

7.4–7.19 Commercial product chapters. This part of the book, consisting of 16 chapters, describes a representative selection of consumer and clinical products, covering the product features and chemistry, with a layout and termi-nology consistent with the theory parts of the book. These chapters are written by product specialists at Abbott, Alere, Beckman, Ortho-Clinical Diagnostics, Phadia, Siemens, and SPD.

PART 8 – IMMUNOASSAY APPLICATIONS OTHER THAN CLINICAL CHEMISTRY

8.1 Immunoassay applications in veterinary diagnostics. This chapter provides comprehensive coverage of immu-noassays used to control and diagnose infectious diseases, and assess metabolic and reproductive status, in veterinary fields. The technologies and science of veterinary immu-noassay tests are explained, covering feline, canine, por-cine, equine, bovine, and avian applications.

8.2 Ligand binding assays in drug development applications. This chapter describes how the pharmaceuti-cal industry applies immunoassays in the different stages of drug research and development, pharmacokinetics, toxi-cology, and clinical studies.

PART 9 – IMMUNOASSAY CLINICAL APPLICATIONS

9.1 Clinical concepts. The principles of clinical test application to diagnosis, patient management and screen-ing, and the consequences for immunoassay design.

9.2–9.23 Clinical chapters. The final part of the book, consisting of 22 chapters, provides an overview of the main clinical fields in which immunoassays are applied, with a description of normal bodily function, relevant clinical dis-orders, and a section on each of the immunoassay analytes likely to be used in that field, in a consistent format. This part of the book describes immunoassay tests in their clini-cal context. Reference intervals are included in these chap-ters for background scientific interest, but must not be applied to any clinical situations as reference intervals vary between methods and patient populations. The clinical subjects are:

ThyroidAdrenal cortexBone metabolismInfertilityIn vitro fertilization and embryo transfer (IVF-ET)Hirsutism and virilization in the femalePregnancyGrowth and growth hormone deficiencyDiabetes mellitusHematologyCardiac markersCancer markersAllergyAutoimmune diseaseGastrointestinal tractHepatitisHuman immunodeficiency virus (HIV)Viral diseases (excluding hepatitis and HIV)Bacterial diseasesParasites and fungiTherapeutic drug monitoringDrugs of abuse