Radioimmunoassay

Radioimmunoassay

Radioimmunoassay

Radioimmunoassay is an extremely sensitive method that can be used for the detection and quantitation of any substance that is antigenic and can be labelled with a radioactive isotope. Basically, the method depends upon the competition between labelled (known) and unlabelled (unknown)antigen for the same antibody. A known amount of labelled antigen, a known amount of specific antibody, and an unknown amount of unlabelled antigen are allowed to react together.

The more of the antigen there is in the test sample, the less chance the labelled antigen has of combining with the limited number of antibody molecules that are available Thus by measuring the quantity of labelled antigen combined with antibody (with isotope counting equipment), a measure of the antigen in the unknown test sample can be obtained. The more labelled antigen combined with antibody, the lower the antigen level in the test sample.

The quantity of isotope-labelled antigen complexing with the antibody varies inversely with the quantity of unlabelled antigen in the test sample. In order to measure the amount of the labelled antigen attached to antibody, it is necessary to separate the antigen-antibody complexes from the mixture. A variety of methods have been developed to separate antibody-bound and unbound labelled antigen. However, the most convenient is the solid phase radioimmunoassay wherein the antibody is linked to an insoluble support, for example, agarose beads. The insoluble complex is then mixed with labelled and unlabelled test antigen. Antibody-bound, labelled antigen can then be separated from free antigen by centrifugation or filtration. By measuring the radioactivity, the percentage of labelled antigen bound to the antibody can be calculated. The concentration of an unknown (unlabelled) antigen can be determined by reference to a standard curve constructed from data obtained by allowing varying amounts of an labelled antigen to complete.

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