Immunoassay

An immunoassay is a specific type of biochemical test that measures the presence or concentration of a substance (referred to as the "analyte") in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed (i.e., measured) using immunoassay methods. In essence, the method depends upon the fact that the analyte in question is known to undergo a unique immune reaction with a second substance, which is used to determine the presence and amount of the analyte. This type of reaction involves the binding of one type of molecule, the antigen, with a second type, the antibody. Immunoassays can be carried out using either the antigen or the antibody in order to test for the other member of the antigen/antibody pair. In other words, the analyte may be either the antigen or the antibody.

For antigen analytes, an antibody that specifically binds to that antigen can frequently be prepared for use as an analytical reagent. When the analyte is a specific antibody its cognate antigen can be used as the analytical reagent. In either case the specificity of the assay depends on the degree to which the analytical reagent is able to bind to its specific binding partner to the exclusion of all other substances that might be present in the sample to be analyzed. In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to permit an accurate measurement. The affinity requirements depend on the particular assay format that is used.

An analyte that has been identified using immunoassay methods is referred to in relevant literature as "immunoreactive." The following excerpt may help illustrate how these terms are used with each other: "By this immunoassay, the concentration and molecular size of immunoreactive gamma 2-MSH in plasma of healthy subjects were examined, and the results were compared with those by competitive enzyme immunoassay. Immunoreactive gamma 2-MSH measured by competitive enzyme immunoassay was a mixture of substances with high molecular weights (100-500 kDa), and its concentration was calculated to be 50-60 pmol/L using gamma 2-MSH (1-12) as standard."

In addition to binding specificity, the other key feature of all immunoassays is a means to produce a measurable signal in response to a specific binding. Historically this was accomplished by measuring a change in some physical characteristic such as light scattering or changes in refractive index. With modern instrumentation such methods are again becoming increasingly popular. Nevertheless most immunoassays today depend on the use of an analytical reagent that is associated with a detectable label. A large variety of labels have been demonstrated including radioactive elements used in radioimmunoassays; enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystallites, gold, silver, and selenium colloidal particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals,and others. Such labels serve for detection and quantitation of binding events either after separating free and bound labeled reagents or by designing the system in such a way that a binding event effects a change in the signal produced by the label. Immunoassays requiring a separation step, often called separation immunoassays or heterogeneous immunoassays, are popular because they are easy to design, but they frequently require multiple steps including careful washing of a surface onto which the labeled reagent has bound. Immunoassays in which the signal is affected by binding can often be run without a separation step. Such assays can frequently be carried out simply by mixing the reagents and sample and making a physical measurement. Such assays are called homogenous immunoassays or less frequently non-separation immunoassays.

Regardless of the method used, interpretation of the signal produced in an immunoassay requires reference to a calibrator that mimics the characteristics of the sample medium. For qualitative assays the calibrators may consist of a negative sample with no analyte and a positive sample having the lowest concentration of the analyte that is considered detectable. Quantitative assays require additional calibrators with known analyte concentrations. Comparison of the assay response of a real sample to the assay responses produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample.

Read more about Immunoassay:  Types