Radioimmunoassay

Radioimmunoassay (RIA) is a laboratory technique used to measure the concentration of antigens, antibodies, hormones, and other biological substances in a sample. It is a highly sensitive and specific method that involves the use of radioisotopes to detect and quantify the amount of a particular substance in a biological fluid such as blood, serum, or urine.

The principle of RIA is based on the competition between a radiolabeled antigen and an unlabeled antigen for a limited number of specific antibody binding sites. The radiolabeled antigen and the unlabeled antigen are mixed together and allowed to bind to the antibody. The amount of radiolabeled antigen that remains bound to the antibody is inversely proportional to the amount of unlabeled antigen present in the sample.

RIA has been used in clinical and research settings to measure a variety of substances, including hormones such as insulin, thyroid hormone, and cortisol; drugs such as digoxin and theophylline; and tumor markers such as prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA). It is a very sensitive method and can detect very low levels of these substances in biological fluids.

Basic principles of radioimmunoassay

Competitive radioimmunoassay (RIA) uses a radioactive tracer, typically I-125, as a labeled antigen (Ag*), which competes with the unlabeled antigen (Ag) in the sample or standard solution of unknown concentration to bind to a limited number of specific binding sites on a quantified antibody (Ab), which is typically a polyclonal antibody. Through appropriate reaction conditions, the labeled and unlabeled antigens compete for binding to the specific binding sites on the antibody, resulting in the formation of Ag*-Ab and Ag-Ab complexes, which are then separated by an appropriate separation method and detected by a gamma counter to obtain counts per minute (cpm) values. Using a computer within the counter, a standard curve can be generated from a series of known standard solutions with concentrations from high to low, which can then be used to calculate the concentration of the unknown sample. The concentration of the unknown sample is inversely proportional to the concentration of Ag-Ab-Ag*.

Non-competitive immunoradioassay

In solid-phase two-step immunoradioassay, two monoclonal antibodies are used to react with the antigen, where one of the monoclonal antibodies is labeled with a radioactive tracer, typically I-125, as the labeled antibody (Ab*), and the other monoclonal antibody is immobilized on a solid-phase support (AbS). The AbS-Ag-Ab* complex, resembling a sandwich, is formed between the antigen in the standard or sample and the two monoclonal antibodies. The unbound labeled antibody can then be easily washed out in the washing stage. The counts per minute (cpm) value is obtained by detecting the labeled antibody with a gamma counter. Using a computer within the counter, a standard curve can be generated from a series of known standard solutions with concentrations from high to low, which can then be used to calculate the concentration of the unknown sample. The concentration of the unknown sample is directly proportional to the concentration of the AbS-Ag-Ab* complex.

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