Antibody Phage Display Methods and Protocols
Since its introduction almost 20 years ago, phage display technology has revolutionized approaches to the analysis of biomedical problems, quickly impacting the fields of immunology, cell biology, biotechnology, pharmacology, and drug discovery. In
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1. Introduction Immunoassays are widely used for the analysis of low molecular weight compounds or haptens in biological samples, and are often the only viable analytical method available. The performance of these assays is critically dependent on the characteristics of the antibody used, and the identification of suitable antibodies is often a major hurdle in assay development. Although the main drive in the development of recombinant antibody technologies has been the desire to develop novel therapeutic antibody activities suitable for clinical applications, the production of anti-hapten antibodies for diagnostic applications can benefit considerably from the recombinant approach. Recombinant antibody technologies provide access to a much greater repertoire of antibody activities than traditional antibody production techniques, allowing selection of antibodies with characteristics that are critical for optimal
Robert Aitken (ed.), Antibody Phage Display, Second Edition, Methods in Molecular Biology, vol. 562 DOI 10.1007/978-1-60327-302-2_7, © Humana Press, a part of Springer Science + Business Media, LLC 2009
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assay performance. They provide a much faster route for identification of the antibody of interest, and the means for continuous supply of the antibody. They also facilitate the modification or manipulation of the antibody for improved compatibility with novel assay platforms. The vast majority of hapten immunoassays use the competitive assay design, in which the analyte competes with a labeled or immobilized version of the target analyte for binding to limited amounts of the antibody. Two-antibody reagent excess or sandwich assays are not commonly used for haptens because haptens are generally too small to bind to two antibodies simultaneously. In a competitive immunoassay, the lower limit of detection that can be achieved is directly influenced by the affinity of the antibody for its antigen. Hence, the higher the affinity of the antibody, the lower is the detection limit that can be theoretically achieved. As the rates of association of antibodies and antigens do not vary enormously, the rate of dissociation or the off-rate of the antigen is particularly important in determining assay performance (1, 2). Antibody specificity is also critical for optimal hapten assay performance. It is often the case that the biological sample being analyzed for hapten contains compounds that are structurally very similar to the target hapten and might also bind to the antibody. These irrelevant compounds might even be in much higher concentration than the target compound, as, for example, the concentration of dehydro-epiandrosterone sulfate in human serum samples for testosterone analysis (3). Alternatively, it may be desirable to measure a family of closely related structures simultaneously, in which case, an antibody with broad specificity would give optimal assay performance. Anti-hapten recombinant antibodies that were first described were derived from preexisting hybridomas; now, these reage
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