ELISA immunoassays include a direct, indirect, sandwich, and competitive variants. In the direct ELISA method, an antigen is immobilized to the surface of a multi-well plate and detected with an antigen-specific antibody directly conjugated to HRP or another detection tag.
For an indirect ELISA assay, as with the direct ELISA method, an antigen is immobilized to the surface of the multi-well plate, but we need a two-step process for detection. The primary antibody binds to the antigen of interest, and then a secondary antibody labeled with a detection reagent is used for quantification. The sandwich ELISA method, the most widely used format, requires an antibody pair where each antibody is specific to a different epitope of the antigen. The first antibody, often called the capture antibody, is coated on the 96 well plate and binds to the analyte of interest. Once the unbound materials in the sample matrix get washed away, the second antibody conjugated with a detection tag quantitates the analyte. Competitive ELISA methods, often referred to as an inhibition ELISA assay or competitive immunoassays, measure the concentration of an antigen by detecting signal interference. We can adapt direct, indirect, or sandwich ELISA to the competitive format. Here, the antigen in the sample competes with a reference antigen pre-coated on a multi-well plate or added in the same plate wells. Consequently, lower concentrations of analyte produce a higher level of the signal. Some competitive ELISA assay, however, utilize a slightly different approach. These kits substitute a labeled antigen for the labeled antibody, but the principle of the assay remains the same.