© 1988 • 726 pp., illus., appendices, bibliography, index
Paper • $79 • ISBN  978-087969314-5

  “[Antibodies aims] to put current immunological methodology into the hands of non-specialists. Happily, Harlow and Lane have succeeded admirably and this manual seems certain also to gain a place on the laboratory bench (too useful to remain on the bookshelf) of a wide variety of biologists—even immunologists. The volume opens with four introductory chapters summarizing key features of the immune response; these provide a succinct overview of the field and are very well written. The scope of the rest of the book includes techniques of immunization and bleeding, monoclonal antibodies and hybridomas, protein labelling, immunoassays (mostly those using solid supports), immunoprecipitation of cellular antigens, immunoblotting and immunoaffinity purification. The methods are presented as easy-to-follow ‘1-2-3 protocols,’ and assume relatively little experience. Rationales for procedures are often presented, which helps to remove some of the mystique that has been associated with immunological methodology.”
      —Nature

Immune Response

An introduction to immunologic principles; Antibody Molecules—review of antibody structure and genetics of immunoglobulin genes; Antibody-Antigen Reactions—review of structure of interaction, affinity, avidity; Antibody Response—how it works; Immunizations—immunogenicity, preparing the antigen, immunization protocols, collecting sera, how to make weak antigens strong; Monoclonal Antibodies—step-by-step production—immunization, developing screening methods, production of hybridomas; Growing Hybridomas—growing and maintaining myeloma and hybridoma cell lines, producing and collecting monoclonal antibodies, methods for drug selection; Storing and Purifying Antibodies; Labeling Antibodies; Cell Staining—constraints, choice of antibody, protocols for staining; Immunoprecipitation—constraints, choice of antibody, protocols; Immunoblotting—constraints, choice of antibody, protocols; Immunoaffinity Purifi-cation—constraints, choice of antibody, protocols; Immunoassay—types of immunoassays, where to start, antibody capture assays, antibody sandwich assays, antigen capture assays, detection, designing immunoassays; Reagents—bacterial cell wall proteins that bind antibodies, anti-immunoglobulin antibodies, proteolytic fragments of antibodies, acetone powders

Appendices

Electrophoresis—one- and two-dimensional gels, alternate buffer systems, silver and Coomassie staining techniques, fluorography, radiography; Protein Techniques—tables, quantitation methods, TCA precipitation; General Information—buffers, stock solutions, detergents; Bacterial Expression—techniques for screening, vectors for bacterial expression

Features

Summary Tables—help you decide strategy for experimental course most suited to your objective; Constraints Indicated—what can and cannot work; Help in Choosing Proper Antibodies for Each Technique; Illustrative Keys—aid in finding proper sequence of techniques; Extensive Advice Based on Authors' Experience; Trouble-shooting—help in avoiding experimental pitfalls and discussion of likely problems and their solution; Cautions—safety notes throughout protocols

Techniques

Detailed Protocols for the Production of Polyclonal and Monoclonal Antibodies—tissue culture supernatants and ascites; Purification of Antibodies—purification of polyclonal and monoclonal antibodies using a broad range of conventional techniques, protein A-based techniques, affinity chromatography; Techniques for Labeling Purified Antibodies with Iodine, Enzymes, Fluoro-chromes, and Biotin—relative advantages and disadvantages for each label; Cell Staining for Tissue Culture Cells, Yeast, and Tissue Sections from the Preparation of Samples Through Detection; Immunoprecipitation—labeling, lysis, and purification of immune complexes with extensive trouble-shooting section; Immunoblotting—from preparation of samples through detection; Immunoaffinity Purification—multiple examples of techniques for preparation of antibody columns and suggested strategies for elution; Immunoassays—theory of immunoassay, advice on choosing the correct method, detailed protocols for six most commonly used methods, including overview of immunoassay design; Special Reagents—protein A and protein G, anti-immunoglobulin antibodies, proteolytic fragments of antibodies, acetone powders