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 detecting infection the presence of antibody against the 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 though 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)), radioisotopes such as I-125 Radioimmunoassay (RIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot.
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 involved a separation step) 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.
Immunoassays can be homogeneous or heterogeneous:
- A heterogeneous immunoassay will require an extra step to remove unbound antibody or antigen from the site, usually using a solid phase reagent.
- Because homogeneous assays do not require this step, they are typically faster and easier to perform.
- Learning Guides from Abbott Laboratories on Immunoassay (English): Part 1 Part 2 Part 3 Part 4 Appendix