A competitive binding assay is based upon the competition of labeled and unlabeled ligand for a limited number of antibody binding sites. Briefly, the free antigen and antibody are incubated to form antigen-antibody complex and then the complex are bound to antigen-coated surface in the assay plate. The unbound antibody-antigen complex is washed off before adding enzyme-linked secondary antibody against the primary antibody. The substrate is then added and the antigen concentration can then be determined by the signal strength elicited by the enzyme-substrate reaction. In this assay, the enzyme-linked secondary antibody compete with the sample antigen which is associated with the primary antibody.