immunoassay

An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody to its antigen. Monoclonal antibodies are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies picked must have a high affinity for the antigen (if there is antigen available, a very high proportion of it must bind to the antibody).

Both the presence of antigen or antibodies can be measured. For instance, when seeking to detect the presence of an infection the concentration of antibody specific to that particular pathogen is measured. For measuring hormones such as insulin, the insulin acts as the antigen.

For numerical results, the response of the fluid being measured must be compared to standards of a known concentration. This is usually done through the plotting of a standard curve on a graph, the position of the curve at response of the unknown is then examined, and so the quantity of the unknown found.

Detecting the quantity of antibody or antigen can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody. The label may consist of an enzyme (see enzyme immunoassay (EIA)), colloidal gold (lateral flow assays), radioisotopes such as I-125 Radioimmunoassay (RIA), magnetic labels (magnetic immunoassay - MIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot.

Immunoassays have a particularly important role in the diagnosis of many Infectious Diseases, including HIV. Immunoassays are just one type of diagnostic HIV test.

Types

Immunoassays can be divided into those that involve labelled reagents and those which involve non-labelled reagents. Those which involve labelled reagents are divided into homogenous and heterogeneous (which require an extra step to remove unbound antibody or antigen from the site, usually using a solid phase reagent) immunoassays. Heterogeneous immunoassays can be competitive or non-competitive.

• In a competitive immunoassay, the antigen in the unknown sample competes with labeled antigen to bind with antibodies. The amount of labeled antigen bound to the antibody site is then measured. In this method, the response will be inversely proportional to the concentration of antigen in the unknown. This is because the greater the response, the less antigen in the unknown was available to compete with the labeled antigen.

• In noncompetitive immunoassays, also referred to as the "sandwich assay," antigen in the unknown is bound to the antibody site, then labeled antibody is bound to the antigen. The amount of labeled antibody on the site is then measured. Unlike the competitive method, the results of the noncompetitive method will be directly proportional to the concentration of the antigen. This is because labeled antibody will not bind if the antigen is not present in the unknown sample.

Because homogeneous assays do not require this step, they are typically faster and easier to perform.

See also

• ELISA
• MELISA
• Lateral flow test
• Magnetic immunoassay

External links

• Learning Guides from Abbott Laboratories on Immunoassay (English): Part 1 Part 2 Part 3 Part 4 Appendix

• MeSH Immunoassay
• Automation of Immunoassays

See also Chapter 5 and 6 in the book "Bioanalytical Chemistry" by Susan R. Mikkelsen