Adapted from Enzyme Immunoassay by Dan Monroe in Analytical Chemistry, Vol. 56, No. 8, July 1984, p920A-931A
EMIT and ELISA are enzyme immmunoassays (EIAs) developed for the measurement of microamounts of substances in test samples, making them useful in routine analytical determinations.
EMIT is an EIA used for rapidly assaying microamounts of drugs and substances in human biological fluids. It is a homogeneous, liquid phase assay that measures haptens for drug, hormone and metabolite determinations. The way an EMIT drug assay works begins with an excess of specific antibodies that bind with the drug being measured. This excess is added to the patient specimen. If molecules of the drug being tested for are present, the molecules bind immediately to the antibody sites. If this occurs, the enzyme-labeled drug is added to the mixture. Molecules of this enzyme-labeled drug fill any antibody binding sites not filled by molecules of the drug in the specimen. Since only free enzyme-labeled drug molecules can act on the substrate, enzyme activity is reduced.The amount of free enzyme left in the mixture determines the amount of substrate that is converted to a colored form from an uncolored one, in a given time period. The color change is then easily measured by a spectrophotometer. There are two basic test principles essential to the EMIT assay. The first is that the enzyme need to remain active after it binds to the compound being measured (hapten), and secondly when the hapten reacts with its specific antibody the enzyme activity of the hapten-enzyme conjugate is either reduced or inhibited. EMIT is unique from other EIAs in that reagent filtration is not required.
Movies demonstrating molecular scale animation of the chemistry of both a positive (561K) and a negative (170K) result of this procedure are available.
ELISA is an EIA used for antigen measurements. It is a heterogeneous, solid phase assay that requires the seperation of reagents. ELISA has two available techniques for antigen measurement, the sandwich tecnique and the competitive technique. The sandwich or double antibody technique begins with an antibody bound to a polystyrene well plus the antigen to be measured (see fig). An enzyme conjugate is then added to the well with bound antigen-antibody or immune complex (see fig). Next a substrate is added to the enzyme conjugate which is bound to the immune complex (see fig). From here if there are changes due to the presence of the enzyme conjugate bound to the immune complex, a positive test or color change will occur (see fig). The antigen competitive inhibition assay begins with an antibody bound to a polystyrene well (see fig) plus a test sample containing an antigen mixture to which an antigen-enzyme conjugate is added (see fig). At this point competitive inhibition occurs between the antigen-enzyme conjugate and an unlabeled antigen, depending on which antigen type is in excess, two different outcomes can follow when binding to a specific antibody occurs. After the formation of an immune complex from an antigen-antibody binding (see fig), the reagents are separated by a washing. Next a substrate is added to the immune complex (see fig). If the antigen-enzyme conjugate is the antigen in excess a color change will occur indicating that the substrate was chemically changed as a result of the enzyme conjugate being bound to the immune complex (see fig). If it is the unlabeled antigen that is in excess there will be little to no change in color because the test sample contains antibody-type-specific antigen.
EMIT and ELISA have many characteristics that separate them from each other. EMIT is mainly used in drug, hormone, and metabolite determinations. It measures haptens which are small molecules while ELISA measures macromolecules such as antigens and antibodies and is used for diagnosing infectious disease and immunoglobulins. EMIT is faster than ELISA but ELISA has greater sensitivity. Both of these EIAs have a long shelf life and can be done by personnel with only minimal training.
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