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Among all the ELISA procedures, the inhibition ELISA—also referred to as the blocking or inhibition ELISA—is arguably the most intricate. It is possible to modify direct, indirect, and sandwich to fit the competitive structure. Through the detection of interference in an expected signal output, the competitive ELISA is primarily used to determine the quantity of an antigen or antibody in a sample. A reference and sample antigen or antigen compete with one another to bind to a restricted quantity of labeled antigen or antibody, respectively. The concentration of small-molecule antigens in intricate sample combinations can be measured with great benefit from the competition ELISA. While reporter-labeled antibody is used in indirect competition ELISA, labeled antigen is incorporated in direct competition ELISA.

Before the sample and labeled antibody are incubated, the microtiter plate is adsorbed with a known antigen standard in the indirect competition ELISA (Format 1). Substrate is added after the sample's unbound antigen and labeled antibody have been removed through washing. An inverse relationship exists between the concentration of antigen in the sample and the amount of labeled antibody bound to solid antigen, which is determined by the amount of antigen present in the sample. Consequently, less solid antigen is bound to the enzyme-labeled antibody and, as a result, the resultant signal is lower the higher the antigen concentration in the test sample.