 The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation.Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound.After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.