Radioimmunoassay

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Radioimmunoassay
Intervention
MeSH D011863

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 among the least expensive methods to perform such measurements. It requires special precautions and licensing, since radioactive substances are used. The unique ability of RIA to measure small molecules can nowadays be achieved in many cases by non-radioactive methods such as ELISA, where the antigen-antibody reaction is measured using colorimetric, such as absorbance, fluorescence intensity or polarization. This, combined with the usual sandwich configuration of ELISA requiring two domains of an analyte to be present to generate a signal, has led to many assays being developed for ELISA instead of RIA or for RIA assays to be replaced by ELISA. However, because of RIA's robustness, historical use as golden standard, consistently reliable results and low reagent cost per test, RIA methods are again becoming popular. RIA is generally simpler to perform and more precise than a bioassay, although it generally takes several days to perform. For comparison, ELISA can usually be performed in a single day. Although ELISA can be configured to use only a single antibody using competition, cases in which only one antibody is available, for instance, very small analytes, typically end up in the domain of the classical "competitive" RIA.

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

Method[edit]

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically 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 standard curve can then be generated which allows the amount of antigen in the patient's serum to be derived. The radioactivity for each of a series of known concentrations of standards are used to derive terms for an equation, often sigmoidal curve fit. Once the terms for the equation are derived, the radioactivity is fed into the equation to obtain concentrations of the unknowns. This function is often integrated into the radioactivity counter, along with functions indicating coefficient of variation and properties of the binding of tracer to antibody. A typical RIA will have somewhere around 50% of tracer bound to antibody in a sample with no analyte. Samples are often run in duplicate to ensure a correct reading.

History[edit]

This technique 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.[1] In 1977, Dr. 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.[2] Yalow and Berson refused to patent the assay, because they felt that it should be freely available to the field of medicine.[2]

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 supernatant (liquid above the centrifuged pellet) is aspirated away from the system. Such pulldown antibodies are typically immobilized to beads, in which case tracer bound to analyte-specific "capture" antibody is measured and the standard curve has declining radioactivity as analyte concentration increases, due to competition with tracer. However, charcoal is frequently used for pulldown, which works equally well, but is far less expensive since no antibody is needed for pulldown and charcoal is inexpensive. When charcoal is used, what is pulled down (by centrifugation) is unbound tracer instead of bound. This is achieved by "blocking" the charcoal with buffer containing dextran and/or bovine serum albumin such that antibody-tracer complexes do not stick to charcoal. When charcoal is used, the standard curve is seen to show increased radioactivity with increased analyte concentration as a result of less tracer bound to the antibody that it must compete against the analyte for binding to. 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.[3] An ultramicro RIA for human TSH was published in BBRC (1975) by Drs. Acebedo, Hayek et al.[4]

References[edit]

  1. ^ Yalow, RS, Berson, SA (1960). "IMMUNOASSAY OF ENDOGENOUS PLASMA INSULIN IN MAN". The Journal of Clinical Investigation 39 (7): 1157–75. doi:10.1172/JCI104130. PMC 441860. PMID 13846364. 
  2. ^ a b Glick S (2011). "Rosalyn Sussman Yalow (1921–2011)". Nature. 474 (7353): 580. doi:10.1038/474580a. PMID 21720355. 
  3. ^ Werner SC, Acebedo G, Radichevich I (1974). "Rapid radioimmunoassay for both T4 and T3 in the same sample of human serum". J. Clin. Endocrinol. Metab. 38 (3): 493–5. doi:10.1210/jcem-38-3-493. PMID 4815178. 
  4. ^ Acebedo G, Hayek A, Klegerman M, Crolla L, Bermes E, Brooks M (1975). "A rapid ultramicro radioimmunoassay for human thyrotropin". Biochem. Biophys. Res. Commun. 65 (2): 449–56. doi:10.1016/S0006-291X(75)80168-9. PMID 1148002. 

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