Radioimmunoassay Radioimmunoassay (RIA), an in vitro nuclear medicine, is a very sensitive technique used to measure concentrations of antigens (for example,

hormone levels in the blood) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of corresponding antigens. Although the RIA technique is extremely sensitive and extremely specific, it requires speciali ed equipment, but remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. !oday it has been supplanted by the "#I$A method, where the antigen%antibody reaction is measured using colorimetric signals instead of a radioactive signal. &owever, because of its robustness, consistent results and low price per test , RIA methods are again becoming popular. It is generally more simple to perform than a bioassay !he RA$! test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy. Contents

' (ethod ) &istory

Method !o perform a radioimmunoassay, a *nown quantity of an antigen is made radioactive, frequently by labeling it with gamma%radioactive isotopes of iodine attached to tyrosine. !his radiolabeled antigen is then mixed with a *nown amount of antibody for that antigen, and as a result, the two chemically bind to one another. !hen, a sample of serum from a patient containing an un*nown quantity of that

same antigen is added. !his causes the unlabeled (or +cold+) antigen from the serum to compete with the radiolabeled antigen (+hot+) for antibody binding sites. As the concentration of unlabelled antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody%bound radiolabeled antigen to free radiolabeled antigen. !he bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. ,sing *nown standards, a binding curve can then be generated which allows the amount of antigen in the patient-s serum to be derived. History It was developed by Rosalyn .alow and $olomon Aaron /erson in the '012s. In '033, Rosalyn $ussman .alow received the 4obel 5ri e in (edicine for the development of the RIA for insulin6 the precise measurement of minute amounts of such a hormone was considered a brea*through in endocrinology. 7ith this technique, separating bound from unbound antigen is crucial. Initially, the method of separation employed was the use of a second +anti%antibody+ directed against the first for precipitation and centrifugation. !he use of charcoal suspension for precipitation was extended but replaced later by 8rs. 7erner and Acebedo at 9olumbia ,niversity for RIA of !: and !;.An ultramicro RIA for human !$& was published in //R9 ('031) by 8rs. Acebedo, &aye* et al.
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 labeled with a radioactive isotope. Basically, the method depends upon the competition between labeled (known) and unlabelled (unknown) antigen for the same antibody. known amount of labeled antigen, a known amount of specific antibody, and an unknown amount of unlabelled antigen are allowed

to

react

together.

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

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