This method is also known as saturation or displacement analysis:

Ab 1 L 1 L*2 Ab. L 1 Ab. L* (12.1)

The analyte to be measured (L) and its labeled version (L*) compete for occupy­ing a limited number of binding sites (Ab). The labeled component may be added some time later. The more (unlabeled) analyte is present in the reaction mixture, the less labeled ligand will be bound. The last step is the separation of bound from free ligand. If a radioactive tracer is used, the method is called radioimmunoassay (RIA). RIAs were first introduced in the 1960s by Yalow and Berson for insulin and by Ekins for thyroxin. The precise measurement of minute amounts of such a hormone was considered a breakthrough in endocrinology; Rosalyn Yalow, a biophysicist, received the Nobel Prize in medicine for the development of RIAs of peptide hormones in 1977.

The accuracy of a RIA is limited by both the competitive nature of the reaction and the efficiency of the separation method applied.

The first, catcher antibody (Ab1) is usually fixed to some solid phase (the wall of the vial, spheres, pearls, etc.). After reaction (12.2), unbound ligand is washed off, and then a second, tracer antibody (Ab2) is added that binds to the ligand molecules present and already fixed. The amount of the bound fraction of Ab2 increases with the ligand (L) concentration. Free Ab2 is washed off as well.

Sandwich methods usually apply excess amounts of the reagents as they are based on the occupation of, rather than competition for, the binding sites. Such methods allow fast, sensitive, and specific measurements. When applying radioac­tive labeling, the method is called immunoradiometric assay (IRMA). The accuracy of IRMA methods is superior to that of RIA and similar to the “alternative” meth­ods that do not apply radioactive tracers.