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 COMPONENTS
There 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
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
PART 4 – RELATED TECHNIQUES
4.1 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
PART 5 – IMMUNOASSAY DEVELOPMENT
5.1 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-
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
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
6.1 Sample collection, including participant prepara-
tion and sample handling. This chapter provides compre-
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
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
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,
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
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
In vitro fertilization and embryo transfer (IVF-ET)
Hirsutism and virilization in the female
Growth and growth hormone deﬁciency
Human immunodeﬁciency virus (HIV)
Viral diseases (excluding hepatitis and HIV)
Parasites and fungi
Therapeutic drug monitoring
Drugs of abuse