Radioimmunoassay

Radioimmunoassay (RIA) is a very sensitive in vitro assay 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, requiring specialized equipment, it remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. Today it has been supplanted by the ELISA method, where the antigen-antibody reaction is measured using colorimetric signals instead of a radioactive signal. However, 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.

The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.

Method
To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" 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. The 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. Using known 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 in the 1950s by Rosalyn Yalow and Solomon Aaron Berson working at the Bronx Veterans Administration Hospital affiliated with the Mount Sinai School of Medicine. In 1977, Rosalyn Sussman Yalow received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise measurement of minute amounts of such a hormone revolutionized the field of endocrinology. By allowing the precise measurement of blood levels of hormones the mechanism of hormone deficiency diseases could be identified, and better treated. Yalow and Berson refused to patent the assay, because they felt that it should be freely available to the field of medicine. Berson would have shared the prize, but he had died before the award was given.

With 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. The use of charcoal suspension for precipitation was extended but replaced later by Drs. Werner and Acebedo at Columbia University for RIA of T3 and T4. An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.