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.