This assay facilitates the immunometric determination of assay-associated reagents including the capture and detection antibodies as well as the analyte (i.e., antigen or antibody). It can precede the development of a sandwich enzyme-linked immunosorbent assay (ELISA) in which optimal antibody concentrations are applied for the quantitative measurement of the antigen. This protocol describes the materials and equipment required for the measurement of chromogenic substrate development; however, it can be adapted for use with chemiluminescent- and fluorescent-labeled reporters.