Quality control of coated antibodies: New, rapid determination of binding affinity

A procedure is described for the determination of the affinity constant bet ween a fluid-phase biotinylated antigen and a solid-phase monoclonal antibo dy. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which grea tly determines the quality of the assay. The free and bound fractions of th e biotinylated antigen were obtained in wells coated with a low level of im mobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antib ody coated wells which captured all the free antigen molecules. These molec ules were quantified using a standard curve established with known concentr ations of biotinylated antigen, also incubated in wells coated with the hig h level of antibody.