This assay is used to detect or quantitate soluble antigens and is most useful when both a specific antibody and milligram quantities of purified or semi-purified antigen are available (Fig. 2.1.2). To detect soluble antigens, plates are coated with antigen and the binding of specific antibody-enzyme conjugates to antigen-coated plates is inhibited by test solutions containing soluble antigen. After incubation with mixtures of the conjugate and inhibitor in antigen-coated wells, unbound conjugate is washed away and substrate is added. The amount of antigen in the test solutions is proportional to the inhibition of substrate hydrolysis and can be quantitated by interpolation onto an inhibition curve generated with serial dilutions of a standard antigen solution.
The direct assay may also be adapted as an indirect assay by substituting specific antibody for specific antibody-enzyme conjugate. The amount of specific antibody bound is then detected using a species-specific or isotype-specific conjugate as a tertiary reactant.
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