Immunoassay's use the basic immunology concept of an antigen binding to its specific antibody, which allows detection of very small quantities of antigens such as proteins, peptides, hormones, or antibody in a fluid sample.
- ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
- Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in blood) by use of antibodies. As such, it can be seen as the inverse of a radio binding assay, which quantifies an antibody by use of corresponding antigens. A known quantity of an antigen is made radioactive, labeled with 125-I. 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 antigen from the serum to compete with the radiolabeled antigen for antibody binding sites. As the concentration of unlabeled 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(unbound) antigen remaining in the supernatant is measured using a gamma counter.
- Immunoradiometric assay (IRMA) is an assay that uses radiolabeled antibodies. It differs from conventional radioimmunoassay (RIA) in that the compound to be measured combines immediately with the radiolabeled antibodies. In IRMA, the antibodies are labeled with radioisotopes which are used to bind antigens present in the specimen. When a positive sample is added to the tubes, radioactively labeled, antibodies bind to the free epitopes of antigens and form an antigen-antibody complex. Unbound labeled antibodies are removed by a second reaction with a solid phase antigen. The amount of radioactive remaining in the solution is direct function of the antigen concentration.