Biochemical Techniques and Instrumentation

3.2.4.2. Indirect ELISA

In indirect ELISA, a labeled secondary antibody is used for the detection of the antigen, that is directly immobilized on the assay plate. The antigen first binds to a primary antibody, which then binds to the labeled secondary antibody. The secondary antibody has specificity for the primary antibody but not for the capture antibody. This assay is therefore not specific for the antigen. The indirect ELISA is the most widely used method.

Indirect Triple Antibody Sandwich (TAS) ELISA

In TAS ELISA, the target antigen is captured by an antibody attached to the microtiter plate. The primary antibody is then added and allowed to bind with the target antigen. The excess antibody is washed away. The capture and primary antibodies should be obtained from different host species (e.g., mouse IgG and rabbit IgG). Then, the secondary antibody with enzyme conjugate is added and allowed to bind indirectly to the antigen.