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“There’s a place….” ( John Lennon and Paul McCartney, ((1963).

First thing in the morning, I walk to the Dee estuary, which lies between England and Wales, to gain a clearer perspective on the day ahead and organize my thoughts. I stroll along a footpath by a hedgerow and cross a track where a steam railway once pounded down a long aban-doned route. I listen to birds singing in the trees and walk onto a cliff top with a view of the Welsh hills across the estuary, which is home to thousands of wading birds in the winter.

As I take my morning constitutional, I often think about immunoassays and how best to present them in this book.

All around me, I can see how our ancestors have been harnessing nature for centuries. My neighbor’s dogs are a product of selective breeding, as are the cows and sheep. The estuary attracts birds because the river was channeled to provide a constant supply of cooling water to a power station, creating large expanses of food-rich tidal mudflats and salt marsh. Seaward, I can see the ferries to Ireland passing a wind farm. I realize that I am continuing this human compulsion to take advantage of nature, even as I walk. During the Spring, I can prompt a vocal response from hidden warblers by playing mp3 versions of their songs through the powerful but tiny speakers on my iPhone.

I find myself wondering: where do immunoassays rank on the scale of human technological achievement, which is all based on our understanding of the natural world?

They are based on a broad and deep scientific knowl-edge in physics, chemistry, and biology. The engineering and biochemistry have become highly complex, and the pace of innovation is faster than in many industries,including automobiles, construction, pharmaceuticals, and protection of the environment. The dual application of cutting-edge biochemistry and engineering places immud -noassay into a select subgroup of highly advanced biotech-nology products.

Edward Jenner discovered the process of vaccination in 1796, exploiting the natural process of immunity for the first time. Immunoassay, as defined in this book, was invented in the 1950s, building on earlier applications of antibodies as reagents to quantify molecules in blood sam-ples. Since antibodies resulted from millions of years of evolution in animals, as part of the body’s defense against parasites, bacteria, viruses, toxins, and other hazards, why not adopt them to provide assays of exquisite specificity and sensitivity? This book explains how it is done.

Immunoassays are remarkable examples of biotechnol-ogy. Using a minute quantity of an immunized animal’s blood, an immunoassay can detect and quantify 1 part in 1,000,000,000,000 of a substance in a complex sample,

without purification. The most sensitive commercial ana-lyzers in hospital laboratories attain this level of specificity and sensitivity routinely. Research immunoassays have detected concentrations as low as 10−21 mol, the concen-tration of a bucketful of chemical dispersed across all the world’s oceans, and immunoassays are now an important tool in the investigation of pollution, a negative conse-quence of man’s interaction with nature.

The core elements of immunoassays have undergone many incremental changes. Immunoassay signals have transitioned from radioactive to enzymatic to chemilumi-nescent, antibodies have progressed from polyclonal to monoclonal to recombinant, and protein assays have con-verted from competitive to immunometric. In just one 10-year period, separation methods evolved from antibody precipitation and centrifugation, through centrifugation of latex particles, to magnetic separation of paramagnetic particles, diverging into microtiter plate washing in some sectors. In the same period, homogeneous assays appeared that do not require a separation at all. Advances in automa-tion have traced another innovation pathway, documented for posterity in the technology sections of the four editions of this book. The diversity of technical approaches in use today indicates that immunoassays are far from being a mature technology.

A rapid pace of change has resulted from pushing the integration of biochemistry and engineering to the limit, made possible by the combined efforts of thousands of engineers, biochemists, chemists, clinical chemists, physi-cists, mathematicians, doctors, programmers, marketers, accountants, managers, and business strategists. The indus-try has provided employment for an estimated 100,000manufacturing workers. What has been the broader eco-nomic impact?

The invention of immunoassay is almost unknown to the general public, in contrast to the breaking of the genetic code in the same decade. But the immunoassay business is considerably larger in dollar terms than all DNA-related businesses combined. At the time of writing, timmunoassay sales (at least $20 billion pa) correspond to 70% of global computer and video game sales revenues.

Although the immunoassay market is dwarfed by the sales of pharmaceuticals, many of the latest biological drugs now transforming medicine are based on advances in monoclonal antibody bioengineering that have their roots in immunoassay research and development, documented since the first edition of this book.

Immunoassays have had a profound influence on medi-cine, explained in the final section (part 9) of the book. The pivotal role of immunoassays in clinical diagnosis and follow-up remains their primary contribution to society, much influencing the effectiveness and cost of medical treatment, which is the largest budgetary expenditure

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category in developed world economies. This edition has been extensively revised to reflect recent changes in clini-cal practice related to immunoassays.

This book is only possible because many experts made time in their busy schedules to write about the theory and applications of the extensive range of immunoassays described, including glimpses at what is on the drawing board for the future. Because of the pace of innovation and the competitive spirit in the immunoassay industry and academia, we have all experienced ups and downs in our careers and had mixed feelings when our peers have made breathtaking leaps forward. But we have each been grateful to play a small part in immunoassay history. I am deeply grateful to the many contributors, past and present, for their commitment to immunoassay education through the pages of this book.

The fourth edition has involved a team of more than 90 authors, with 12 new chapters and significant revisions made to most of the existing chapters. Some of the original book authors have retired. Their chapters have been revised in this edition by new leaders in their fields, and in some chapters, we have brought together experts from dif-ferent institutions because of their complementary knowl-edge and experience. In a handful of cases, chapters written by intellectual giants of the field that received great acclaim when the book was first published have been retained with minimal changes because of their high reputation and timeless content. These chapters are still used for refer-ence as breathtaking new technologies, such as digital ELISA, are developed for the future. In total, 124 authors, past and present, contributed material to this edition.

There has been no let up in the pace of innovation since the previous edition of The Immunoassay Handbook was published in 2005. Many believed that the publication of the human genome would signal the end of immunoassays as the dominant medical research tool, as researchers prepared to switch from analysis of proteins to genes. I thought that the third edition might be the last. But the surprising finding that there are only about 25,000 genes in the human genome has brought the spotlight back onto immunoassays, as the genes code for multiple variants of proteins. In the field of proteomics, immunoassays can help to trace metabolic pathways and control mechanisms in cells, to understand how genes can control so many intricate biological functions and how gene expression is controlled. In this edition of The Immunoassay Handbook, the fields of immunohistochemistry and immunocyto-chemistry have been added, as they are closely related to immunoassay in life science research.

A tool that threatened to topple immunoassay from its high perch was mass spectrometry, which has been refined to the point where it can analyze protein mixtures with great specificity, using a more direct approach than immu-noassay. But for generating an assay that can be used to screen large numbers of samples at low cost with compara-tively cheap equipment, immunoassay continues to reign supreme.

What this book shows is that when industry and aca-demia work together there is no end to the sparkling bril-liance of the innovation that can be achieved. Again and again, incremental improvements have been made that moved the field forward. Much of the time, the innovation

was 1% inspiration and 99% perspiration, with large teams working together as immunoassay systems became ever more complex. The last immunoassay development pro-gram I worked on involved three large development teams in the USA and the UK, with a budget of $250 million. Only someone who has worked on a new product intro-duction can understand how demanding the process is and how deep the satisfaction when the first customers express their delight with the outcome.

If you enjoy reading about the application of science, you have come to the right place. Immunoassay provides a master class in the life cycle of a technology business that is still unfolding.

Just a brief note on nomenclature. In The Immunoassay Handbook, assays are described either as competitive or immu-nometric most of the time. I like the term “competitive” because it has a strong descriptive element that helps new-comers to remember how these assays work. However, it is not universally accepted as a scientific description, as some-times the tracer is added after the antibody has had time to bind to analyte in the sample. I also have a bias toward “immunometric” because the somewhat negative connota-tions of “noncompetitive” or “reagent excess” belie their superior performance compared to their competitive coun-terparts. I also think the popular term for the immunomet-ric format “sandwich assay” is potentially misleading, although it is descriptive, as competitive immunoassays may also have three or more layers of molecules bound together. Having justified the use of these terms in the book, I must also point out that although most immunometric assays are also noncompetitive, the term immunometric was origi-nally introduced to describe any immunoassay that uses a labeled antibody, and there are some assays that are com-petitive and use labeled antibodies. In these cases, the assays are described in the book as competitive rather than immu-nometric. I hope the experts will forgive these attempts to simplify and unify nomenclature in order to standardize the book across a range of fields that have sometimes developed independently. Many times in the book you will see the term sandwich alongside immunometric because in some applications it is much more commonly used.

ELISA is a term used in its correct context in the book. It describes solid-phase immunoassays with enzyme-based labels. This is a subset of immunoassays that became prominent when radioactive labels fell out of favor. But the majority of immunoassays performed on hospital analyzers now have chemiluminescent labels, because of their improved reagent stability and the need for signal genera-tion to occur quickly. So ELISA should not be used as a generic term for all immunoassays.

This book is packed with information about immunoas-says. The contributing authors have condensed an amaz-ing amount of information into their chapters without making the subject indigestible. They have taken the time to tell the story of immunoassay in an accessible way, so that readers at all levels can be let into its secrets. The col-laboration between the experts writing in this book contin-ues the spirit that began when Rosalyn Yalow and Solomon Berson, who refused to patent their invention, trained the founders of the immunoassay industry, leading to its rapid growth (see FOREWORD). The authors of this book, from industry, medicine and academia, have set a wonderful

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example by sacrificing their time and passing on their knowledge and experience in a digestible form.

I hope you find this book a welcome source of informa-tion and insight and discover for yourself something new and exciting in the field of immunoassay and its many applications. If you do, you will be continuing a long tradi-tion of scientific fascination with this intriguing subject.

As I return from the estuary and climb the gentle incline to my house, I reflect on 25 satisfying years working on this book, from the first edition to the fourth, as the immunoas-say field has taken me on an exciting and rewarding journey.

David Wild, david@davidwild.net