Epitope mapping from real time kinetic studies - role of crosslinked disulphides and incidental interacting regions in affinity measurements: study with human chorionic gonadotropin and monoclonal antibodies.

Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (alpha 5--14 and alpha 55--62). The association rate constant (k+1) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope- paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region alpha 35--47 and alpha 60--84. The IIRs contribute significantly towards the KA of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb, KA is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.

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.

Real-time kinetic analysis of hCG-monoclonal antibody interaction using radiolabeled hCG probe: presence of two forms of Ag-mAb complex as revealed by solid phase dissociation studies.

Real-time kinetics of ligand-ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand-ligate interaction can be studied using nitrocellulose as a solid support.