The immunoassay is an analytical technique for measuring a targeted antigen, which is also referred to as an analyte. A critical component of the immunoassay is the antibody, which binds a specific antigen. The binding of antibody and targeted antigen forms the basis for immunoassay, and numerous formats have been devised which permit visual or instrumental measurements of this reaction. Antibodies are commonly employed to detect organisms by binding to antigens, usually proteins or polysaccharides, on the surface or "coats" of organisms. The analysis is usually performed in a complex matrix without the need for extensive sample cleanup. Many immunoassays are now readily available from commercial sources, permitting laboratories to rapidly develop in-house immunochemical analytical capability without lengthy antibody preparation. Some of the more widely used formats are as follows: (1)
Radioimmunoassays (RIA) Radiolabelled antigen is quantitatively added to antibody along with various concentrations of unlabeled antigen. The unlabeled antigen competes with the radiolabelled antigen for binding to the antibody. Thus, the higher the concentration of unlabeled antigen in the sample, the lower the level of radiolabelled antigen-antibody complexes. The unbound antigens are removed prior to determining the amount of radiolabelled antigen-antibody formed. A standard curve is then constructed showing the effect of known amount of unlabeled antigen on the amount of radiolabelled antigen-antibody formed. It is now possible to determine the amount of an unknown, unlabeled antigen present in a sample by determining where the value is located on the standard curve [Garvey 1977]. Alternatively, radiolabelled antibody is employed.
(2)
Fluorescent Immunoassays (FIA) Utilization of fluorescent-labeled antibodies to detect bacterial antigens was introduced by Coons et al. [1941 and 1942]. Various FIA techniques have now evolved. These are referred to as: (1) direct FIA, to detect antigen (cell-bound) using fluorescent antibody; (2) indirect FIA, to detect antigen (cell-bound) using antibody and fluorescent antigamma globulin antibody; and (3) indirect FIA, to detect serum antibody using antigen, serum, and fluorescent antibody. Various fluorescent dyes, such as fluorescein, fluorescein isothiocyanate, and rhodamine isothiocyanate, may be employed. A fluorescent microscope is used to evaluate the samples and to count the number of fluorescent organisms [Garvey et al. 1977]. FIA is used to detect viruses and microorganisms.
(3)
Enzyme Immunoassays (EIA) The binding of an antibody or antigen to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), is the basis of EIA techniques. Enzymatic activity, in the presence of a chromogen, results in a colored end-product. Quantitation is performed using a spectrophotometer [Monroe 1984]. Many, if not most, commercially available EIAs are enzyme-linked immunosorbent assays (ELISAs). In this competitive-binding EIA, the antibody is coated onto the surfaces of test tubes, or wells of a microtiter plate, and antigencontaining samples and enzyme-linked antigen are added, resulting in a color change. The more intense the color, the less antigen or analyte are present in the sample. ELISAs can be qualitative or quantitative. A standard curve must be generated if quantitative results are
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NIOSH Manual of Analytical Methods
