This ELISA kit is an indirect competitive enzyme immunoassay. Its main principle is that the unlabeled antibody is incubated in the presence of its antigen (sample). These bound antibody/antigen complexes are then added to an antigen-coated well. The plate is washed, so unbound antibody is removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available to bind to the antigen in the well, hence "competition".) The secondary antibody, specific to the primary antibody, is added. This secondary antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. The reaction is stopped to prevent eventual saturation of the signal.