Selection of antibodies for immunoaffinity chromatography

A broad choice is available in immunoaffinity chromatography, of using polyclonal antibodies or monoclonal antibodies, and this chapter will concentrate mainly on the latter, although many points are common to both. Another broad distinction can be made between immobilized antigens (which may include immunoglobulins) used to prepare purified antibodies, and immobilized antibodies used to prepare pure antigens; again, this chapter will concentrate mainly on the latter. Until recently there was little rational choice possible in antibody selection for immunoaffinity chromatography-one simply had to use the individual animal species bleeds as available and hope for the best (1). The advent of monoclonal antibodies has, in principle, broadened our choice (2), but also has shown up the need for principles and techniques to produce, select, and use antibodies on a rational basis for immunoaffinity work (3, 4). Although this chapter will deal mainly with the selection process (5), it is also necessary to consider and control other aspects, such as immunization (6), fusion and cloning, preparation of solid-phase antigens and antibodies, and selection and testing of eluants (7). Unfortunately, these aspects are far from being exact sciences, and so a considerable degree of empirical testing is still needed (8). Much of the pain and tedium can be removed from such repetitive empirical testing by the sensible choice of automated or semiautomated assay techniques (e.g., enzyme-linked immunosorbent assay [ELISA], radioimmunoassay [RIA], immunoradiometric assay [IRMA], and the like) (9, 10), and assay development thus rests at the core of successful antibody selection for immunoaffinity work.