The central event of competitive ELISA is a competitive binding process executed by original antigen (sample antigen) and add-in antigen. The procedures of competitive ELISA are different in some respects compared with Indirect ELISA, Sandwich ELISA and Direct ELISA.
1.Primary antibody (unlabeled) is incubated with sample antigen.
2.Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the same antigen.
3.Unbound antibody is removed by washing the plate. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
4.The secondary antibody that is specific to the primary antibody and conjugated with an enzyme is added.
5.A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
6.For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal.