When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or antibodies, the technique of detection of the antigen–antibody complex is called as radio-immunoassay (RIA). RIA was first described in 1960 for mea-surement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the Veterans Administration Hospital in New York. Yalow was awarded the 1977 Nobel Prize for Medicine for the development of the RIA for peptide hor-mones, but because of his untimely death in 1972, Berson could not share the award.
The classical RIA methods are based on the principle of competitive binding. In this method, unlabeled antigen competes with radiolabeled antigen for binding to antibody with the appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are incubated with the corresponding antibody, the amount of free (not bound to antibody) radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the mixture.
In the test, mixtures of known variable amounts of cold antigen and fixed amounts of labeled antigen and mixtures of samples with unknown concentrations of antigen with identi-cal amounts of labeled antigen are prepared in the first step. Identical amounts of antibody are added to the mixtures. Antigen–antibody complexes are precipitated either by cross-linking with a second antibody or by means of the addition of reagents that promote the precipitation of antigen–antibody complexes. Counting radioactivity in the precipitates allows the determination of the amount of radiolabeled antigen pre-cipitated with the antibody. A standard curve is constructed by plotting the percentage of antibody-bound radiolabeled anti-gen against known concentrations of a standardized unlabeled antigen, and the concentrations of antigen in patient samples are extrapolated from that curve. The extremely high sensitivity of RIA is its major advantage:
The main drawbacks of the RIA include: (a) the cost of equip-ment and reagents, (b) short shelf-life of radiolabeled com-pounds, and (c) the problems associated with the disposal of radioactive waste.