Enzyme Mediated Immunoassay

T. T. Ngo and H. M. Lenhoff Department of Developmental and Cell Biology University of California, Irvine, CA 92717 In 1959, Yalow and Berson used insulin labeled with radioactive iodine to develop a quantitative immunological method for determining the amount of insulin in human plasma. Their method depends upon ~ competition between insulin labeled with radioactive iodine (II 1) and unlabeled insulin from plasma for a fixed and limited number of specific binding sites on the antibody to insulin. The amount of the labeled insulin bound to the antibody is inversely proportional to the amount of insulin in the plasma sample. Their method, which is so elegantly simple in concept, is made possible by the ability to detect with ease extremely low levels of radioactivity, and by the exquisite specificity of an antibody capable of specifically binding the analyte. Such a combination of sensitivity and specificity is the basis of this versatile analytical tool called radioimmunoassay (RIA). Twelve years later, Engvall and Perlmann (1971) and Van Weemen and Schuurs (1971) independently introduced the use of enzymes as another category of sensitive and even more versatile labels for use in immunoassays. Engvall and Perlmann (l971) coined the term ELISA, which stands for Enzyme Linked Immunosorbent Assay.

The purpose of this book is to focus attention on some of these ideas and concepts. In doing so, it has captured a glimpse of the past and it attempts a projection of the future, but mostly it reveals an overview of the field as it exists as the present time. It aims to serve to spawn further growth in ideas and encourage applications to increasingly broader segments of both clinical and general analytical chemistry fields.

The basis of all immunoassays is the interaction of antibodies with antigens. The most widely used immunoassay technique is radioimmunoassay (RIA) which was first developed by Yalow and Berson in 1959. The principle of RIA is elegantly simple. It utilizes a competitve binding reaction between analytes and a radio-labeled analog of the analytes (the tracer) for anti-analyte antibodies. In addition to its exquisite specificity, extraordinary sensitivity, good accuracy and precision, ease and rapidity of assay and simplicity of assay development, the applicability of RIA to a wide variety of substances has made it one of the most powerful and versatile analytical methods of the 20th century and beyond. Millions of RIA's are being performed annually on clinical, biological and environmental samples in licensed laboratories. In order to expand the use of RIA beyond the confines of these laboratories to areas like physician's offices, patients' homes, economically less developed countries, agricultural fields, large scale and continuing screening tests for infectious diseases, it has become necessary to develop non-isotopic labels. Indeed the last fifteen years have seen the development of a great number of ingenious non-isotopic labels in immunoassay so that a whole new industry capitalizing on the potential market for non isotopic immunoassays has appeared. It is the purpose of this volume to present in depth, state-of-the-art reviews on techniques used in non-isotopic immunoassays. Topics covered include: (1) Enzyme-labeled immunoassay; (2) Luminescene immunoassay; (3) Immunoassay at liquid-solid interface; (4) Membrane immunoassay and (5) "Particle"-mediated immunoassay.

Each chapter of this book aims to explore the basic physical and chemical principles involved in the immunoassay techniques discussed. The book also looks at the optimization and limitations of methodology and concludes with a brief overview of the application of the performance of the technology.

Immunochemistry of Solid-Phase Immunoassay fills a niché in the field of immunoassay and immunology. Although solid-phase immunoassay constitutes a major technology in biology and medicine, there is no comprehensive source devoted to the immunochemical principles involved. As a result, this book will benefit students, technicians, and researchers who use this technology, as well as immunodiagnostic and biotech companies who develop the technology. The book is not a methods manual; instead, it incorporates the concepts, data, and opinions of more than 25 investigators working in this field. Topics discussed include: the chemistry of solid-phases, the behavior or antibodies and antigens on solid phases, membrane solid-phases, reaction kinetics, antigen quantitation, enzyme systems, photophysics, immunochemical considerations in data analyses, multianalyte assays and occupancy concepts, antibody quantitation, streptavidin, a review of data analysis software, and solid-phase peptide immunoassay.

Containing updated and new information on advanced technology - including micro and nanoscale immunoassays - this text provides a mix of practical information coupled with a review of clincal applications and practical examples.

This book shows the various sandwich assays that are constructed from recognition molecules, such as antibodies, oligonucleotide sequences and aptamers, developed as a result of nano- and biotechnology advances. It consists of ten chapters presenting interesting examples of these assays, organized according to the type of analytic methods (colorimetric, fluorescence, electrochemical, etc.) and detected objects (protein, nucleic acid, small-molecule, ion, etc.). It also includes a chapter discussing the introduction of sandwich assays as biosensors for the detection of a range of targets. It is an interesting and useful resource for a wide readership in various fields of chemical science and nanotechnology.

This unique reference provides a pragmatic approach to the development of successful commercial immunodiagnostic products based on enzyme immunoessay technology. Presenting both the basic and applied principles, Enzyme Immunoassays gathers information on all aspects of this process, from the initial conceptualization to the introduction of the product to the market.