Radioimmunoassay (RIA) and enzyme immunoassay (EIA), also known as ELISA (enzyme-linked immunosorbent assay) (Fig. 2.28), are now used very frequently to test for antigens and antibodies. All absorbency tests involve the fixation of antigens or antibodies to a plastic surface. The lower detection limit is a few nanograms. This method forms the basis of modern hepatitis serology, HIV tests, and tests for autoantibodies, lymphokines, cyto-kines, etc. All of these assays can be performed in a direct form (different sandwich combinations of antigen, antibody and anti-antibody, Fig. 2.27)
Fig. 2.27 For solid phase tests both the antigen and antibody are bound to a solid phase (e.g., plastic surface). Various methods are then used to detect any interaction between the antigen and antibody. In the direct test (a) an immobilized, unknown, antigen can be detected using a fluorescent-labeled antibody. If the immobilized antigen is known, this test method can also be used to detect an antibody bound to the antigen. In the sandwich method (b) a known antibody is immobilized. Detection of antibody-antigen binding is then performed using a second, labeled antibody which interacts with the antigen at a different site. The capture method (c) can be used to detect any antigen, for instance IgM antibodies. First, anti-IgM antibodies are immobilized, then serum containing IgM is added to them. The bound IgM can then bind a foreign antigen (e.g., a virus). The detection procedure next makes use of either the labeled foreign antigen or a specific, additionally labeled, antibody which binds to the bound antigen but not to the plastic bound antibody. In the competition or competitive inhibition test (d) antibodies are immobilized, and labeled antigens are then bound to them. An unlabeled (unknown) antigen is added, which competes with the labeled antigen. The level of interaction between the antibody and the unknown antigen is then determined by measuring attenuation of the signal. ►
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or as competition assays. Fig. 2.28 illustrates the quantitative IgE assay, Fig. 2.29 the procedure for detection of specific IgE in patient sera. Analogous procedures are used to detect specific antibody-binding cells or cytokine-releas-ing T cells (Fig. 2.30).
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