Determination of binding constants of diabodies directed against prostate‐specific antigen using electrochemiluminescence‐based immunoassays

Two diabody molecules directed against the prostate‐specific antigen (PSA) were generated from a combinatorial phage display library. The C‐termini of diabodies incorporated the FLAG® peptide epitope (P008D diabody) or the myc epitope (P001D). Both diabodies have the same antigen‐binding site. Equilibrium‐binding constants of these molecules were determined in two immunoassays using the ORIGEN® detection system based on electrochemiluminescence. The binding of diabodies to biotinylated PSA was detected with either polyclonal anti‐mouse Fab F(ab′)2 or a monoclonal antibody directed against the FLAG epitope. Both detecting antibody preparations were covalently labeled with a ruthenium (II) tris (bipyridyl) moiety, Ru(bpy)32+, which allows quantification via an electrochemically triggered light reaction in an ORIGEN analyzer. The binding constants obtained by Scatchard analysis of non‐linear curve fitting calculations from electrochemiluminescence immunoassays were compared with data derived from kinetic‐binding studies using the BIAcore® technology based on surface plasmon resonance. Depending on the detecting antibody, the dissociation constants KD determined at equilibrium with the ORIGEN technology are between 0.1 and 0.4 nM for both diabodies. From the kinetic constants kon and koff measured with the BIAcore instrument KD was calculated to be 0.2 nM for P001D and 0.6 nM for P008D. It is concluded that these two very different methods generate comparable affinity data for the diabodies.