Radioimmunoassay (RIA) was introduced into medical practice still before the enzyme immunoassay. At first, it had many practical applications. By means of RIA, it became possible to test a large number of samples in a very short period of time using very small amounts of reagents.
- Nevertheless, being more difficult in handling, RIA required high-cost equipment, radioactive isotopes and special safety measures.
- Thus, it is being ousted by EIA. Now RIA is used predominantly to determine the quantity of antigens or haptens that can be radioactively labelled.
- These antigens are usually the substances of low molecular weight (drugs, chemicals, toxins, etc.)
- This variation of RIA is based on the competition for specific antibody between the labelled and non-labelled antigen of unknown concentration (competitive RIA).
- In this case, the specific antibodies are adsorbed on the solid phase (e.g. plastic globular particles). The known quantity of labelled antigen is added to Ab-coated particles.
- Usually, 125I or 131I isotopes are used here as a radioactive tag. After antigen–antibody binding, the immune complexes are formed.
- Then the clinical samples containing the unknown concentrations of an Ag are introduced into the system.
- The unlabeled antigens start to replace the labelled antigens in Ab-Ag-complex. This replacement is proportional to the unknown antigenic concentration in the sample.
- As a result, the labelled antigens are released from the solid phase to the surrounding liquid medium.
- After the separation of solid and liquid phases of the reaction, the amount of radioactivity is measured using a counter for radioactivity.
- The concentration of the unknown (non-labelled) antigen or hapten is determined by comparing the results with those obtained with several concentrations of a pre-determined standard antigen.
- This method can detect less than 1 ng/ml of an antigen.
- RIA is an extremely sensitive method applied to the assay of hormones or drugs in biological fluids. A specialized RIA, the radioallergosorbent test (RAST), is used to estimate the quantity of serum IgE antibodies that react with a known allergen (antigen).