This text is a summary of basic principles and techniques and is dedicated to all those students who have been told by their mentors, "Go forth and do two-dimensional gels and have the results on my desk tomorrow. " No attempt has been made in this text to provide exhaustive lists of references related to basic principles or techniques or to list every com pany or supplier involved in this area of research. Nevertheless, it is hoped that sufficient information is given to help a new investigator or student appreciate the complexities but develop sufficient expertise to carry out these techniques successfully. The discussions are designed to instill in basic science and clinical investigators of all levels of expertise an appreciation of the power of combining a variety of techniques as well as to provide basic insight into the theories, complexities, and problems frequently encountered with electrophoretic and immunochemical meth ods. Bonnie S. Dunbar Houston v Acknowledgments I wish to thank my students and staff for their patience and support throughout the preparation of this text. I would like to acknowledge my appreciation for my extensive discussions with Dr. David Sammons (Uni versity of Arizona) and to Dr. N. L. and Dr. N. G. Anderson and their colleagues (Argonne National Laboratory) for their invaluable advice and suggestions in this area over the years. I thank my research assistant, Ms.

Immunological Methods a compendium of basic research techniques being used in one of the largest immunology research institutes, the Basel Institute for Immunology, with particular emphasis given to new methodology. The procedures have been described by individuals judged to be highly expert in their specialties. In many instances the methods developed or adapted to unique uses by the contributors have not previously been described in detail. The book contains 34 chapters covering techniques for detection, isolation, and purification of antibodies (including dansylation, two-dimensional chromatography, isoelectric focusing, polyacrylamide gel electrophoresis, and isotachophoresis); measurement of equilibrium constants (equilibrium dialysis, filtration, and sedimentation); and isotope and fluorescent labeling and detection of cell-surface components. Techniques such as isotope laboratory maintenance; chemical modification of proteins, haptens, and solid supports, and haptenation of viable biological carriers; production of antisera against allotypes and histocompatibility antigens and production of antibody with clonai dominance; histocompatibility and MLR testing; and cell separation by haptenated gels and by velocity sedimentation of rosette-forming cells are also discussed. Other chapters cover detection of antibody-secreting and alloantigen-binding cells; immune responses in vitro and their analysis by limiting dilution; production of T-cell factors; hybridoma production by cell fusion; maintenance of cell lines and cloning in semisolid media; and the mathematical analysis of immunological data.

This text is a summary of basic principles and techniques and is dedicated to all those students who have been told by their mentors, "Go forth and do two-dimensional gels and have the results on my desk tomorrow. " No attempt has been made in this text to provide exhaustive lists of references related to basic principles or techniques or to list every com pany or supplier involved in this area of research. Nevertheless, it is hoped that sufficient information is given to help a new investigator or student appreciate the complexities but develop sufficient expertise to carry out these techniques successfully. The discussions are designed to instill in basic science and clinical investigators of all levels of expertise an appreciation of the power of combining a variety of techniques as well as to provide basic insight into the theories, complexities, and problems frequently encountered with electrophoretic and immunochemical meth ods. Bonnie S. Dunbar Houston v Acknowledgments I wish to thank my students and staff for their patience and support throughout the preparation of this text. I would like to acknowledge my appreciation for my extensive discussions with Dr. David Sammons (Uni versity of Arizona) and to Dr. N. L. and Dr. N. G. Anderson and their colleagues (Argonne National Laboratory) for their invaluable advice and suggestions in this area over the years. I thank my research assistant, Ms.

Immunoassays are among the most powerful and sensitive technologies now available for patient diagnosis and monitoring. This book is an indispensable guide to information on the theory and practice of immunoassays. It discusses the scientific basis of these technologies in a logical, organized, and heuristic manner and provides protocols for specific assays. The contents of this unique book are balanced among theory, practical issues, quality control, automation, and subspecialty areas, making it ideal for health science students, laboratory scientists, and clinicians. - Presents up-to-date information - Provides extensive cross-referencing - Covers theory and practice in full detail - Written by leading authorities

The book appeared in two previous Slovak editions for university students in Czechoslovakia. This edition presents a completely new version updated according to recent advances not only in immunochemistry and essential immunology but also in molecular biology, biochemistry and molecular genetics. The scope of the book is considerable since the goal was to cover the field of immunochemistry from the widest point of view including both the topic and methods of contemporary immunochemistry. Each chapter provides basic information on a specific subtopic, clearly and understandab ly, and presents principles of individual immunochemical methods. I am confident that the book will fill the gap between the books on essential immunology and highly specialised books on individual areas of immunoche mistry (e. g. on antibodies, antigens, numerous immunochemical techniques, etc. ). It may also prove useful for beginning investigators from different biological and medical fields as it supplies basic information needed for solving their scientific problems by immunochemical approaches. I do hope that readers will find the text stimulatory and pleasury to read. I wish to thank all colleagues and friends for supplying their own results, suggestions and for their encouraging comments. My thanks go also to the editors and publishers for their valuable contribution to the preparation of the book. 1 Introduction The term immunochemistry was coined by the Swedish chemist ARRHENIUS who used it for the first time in his lectures in 1907.

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.

Proteins are the functional units of the cellular machinery and they provide significant information regarding the molecular basis of health and disease. Therefore, techniques to separate and isolate the various proteins are critical to studying and understanding their functional characteristics. One of the widely used techniques for this purpose is electrophoresis. In Protein Electrophoresis: Methods and Protocols, contributions from experts in the field have been collected in order to provide practical guidelines to this complex study. Each chapter outlines a specific electrophoretic variant in detail so that laboratory scientists may perform a technique new to their lab without difficulty. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Protein Electrophoresis: Methods and Protocols seeks to serve laboratory scientists with well-honed, detailed methodologies in an effort to further our knowledge of this essential field.

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