Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay

Abstract

Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant ( K aff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure K aff by solid-phase EIA. Based upon the Law of Mass Action and using serial dilutions of both antigen (coating the plate) and antibody, K aff has been measured by EIA. A microtiter plate was coated with antigen (Ag) and then incubated with monoclonal antibody (Ab). The plate was sequentially incubated with a second enzyme-antibody conjugate (EAC) and with the enzyme substrate. The amount of Ab adherent to Ag on the plate [Ag Ab] and [Ag 2 Ab] was reflected by the enzyme product measured by OD. The use of serial dilutions of Ab resulted in a sigmoid curve of OD versus logarithm of total Ab added to the well. Comparison of the OD at the upper plateau (OD-100) for different antibodies was a reflection of the relative number of epitopes on the Ag that were identified by the different antibodies, provided excessive EAC was used. [Ab] t and [Ab′] t were the measurable total antibody concentrations in the wells at OD-50 and OD-50′ for plates coated with [Ag] and [Ag′], respectively. [Ag] and [Ag′] were not true antigen concentrations, but were a measure of antigen density on the plate. For [ Ag′] = [ Ag]/2, K aff = 1 2 (2[ Ab′] t − [ Ab] t ) . Using five different anti-CEA antibodies and different proportions of CEA in the coating solution, K aff was measured. K aff determined by EIA correlated well with K aff measured by soluble phase inhibition assay. This EIA method of estimation of K aff is simple, rapid, and reliable.