Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore.

Kinetic studies of macromolecular ligant-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1:1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chlorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation in insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a sample biomolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms.

Thermodynamics of hCG--monoclonal antibody interaction: an analysis of real time kinetics data obtained using radiolabeled hCG probe.

A thermodynamic analysis of the interaction of 125I-labeled human chorionic gonadotropin (IhCG) with two of its monoclonal antibodies (MAbs) was carried out. The dissociation profile of IhCG-MAb complex conforms to a two-step model. vant Hoff enthalpies were calculated with the K(A) (equilibrium constant) values obtained from dissociation at different temperatures. Free energy and entropy changes were calculated using the standard equations. DeltaH values for one of the MAbs, viz. VM7 were favorable at temperatures beyond 30 degrees C. Interestingly, the DeltaS values were also favorable at all temperatures. In the case of MAb VM4a, however, the interaction throughout the temperature range was driven by large favorable entropic contributions, indicating the importance of hydrophobic interaction in the binding of this MAb to hCG. The energetics of the interaction of these two monoclonals with hCG is discussed.