Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor.

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.