Immunoassay radiolabeling procedures

The most common, but by no means the only or even the most promising, immunochemical assay for small molecules is radioimmunoassay (R1A). As an overview, an immunoassay involves chemically attaching the small molecule of interest (or a derivative of it) to a carrier protein and raising specific antibody titers to it in the serum of an animal. Very dilute antibody solutions are then used to bind the small molecule which has been radiolabeled. The competition of varying known concentrations of unlabeled material is measured and the resulting standard curve used to determine unknown concentrations (Table 1). The steps leading to the development of an R1A are outlined below followed by a description of other immunochemical procedures and an analysis of the attributes and limitations of immunoassay. 

Antigens, haptens, and antibodies radiolabeled with or are commonly used as tracers in immunoassay. These nuclides can be introduced directly into functional groups normally present in proteins and other macromolecules or into suitable derivatives that can be synthesized by a variety of chemical procedures. The most widely used iodination methods have been direct chemical or enzymic substitution of hydrogen in tyrosine or related groups using chloramine-T or lactoperoxidase, respectively. These methods are described in separate chapters in this volume. 

All immunoassay procedures are based on the original discovery by Berson and Yalow that low concentrations of antibodies to the antigen hormone insulin could be detected radiochemically by their ability to bind radiolabeled ( I) insulin. The determination of unknown concentrations of antigen, then, is based on the fact that radiolabeled antigen and unlabeled antigen (from the sample or a standard) compete physiochemically for the binding sites on the antibodies (radioimmunoassay, RIA). This is illustrated in Figure 24.5. 

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