ANALYSIS OF BINDING OF MONOCLONAL-ANTIBODY TO A MALARIAL PEPTIDE BY SURFACE-PLASMON RESONANCE BIOSENSOR AND INTEGRATED RATE-EQUATIONS

Using biosensor technology and integrated rate equations, we have deve loped procedures to determine the kinetic parameters and equilibrium a ffinity constant of Ag-Ab interactions. The Ag used in these studies w as a peptide that represents the major B cell epitope of the circumspo rozoite protein of Plasmodium falciparum, a promising malaria vaccine candidate Ag. Measurements of association and dissociation rate consta nts of this peptide with the mAb 2A10 were determined by fitting integ rated rate equations to binding data obtained with a BIAcore surface p lasmon-resonance biosensor. We examined whether accurate estimates of initial velocity and final equilibrium levels of binding of Ab to pept ides can be obtained using these methods, and whether kinetic rates an d equilibrium constants obtained with systematic variation of the expe rimental parameters conform to a simple bimolecular model of binding. We found that initial velocity was approximately first order with resp ect to Ab concentration. When we used a series of four sensor cells wi th different peptides loads, however, we found that the initial veloci ty of binding appeared to be nearly independent of peptide concentrati on. Equilibrium analyses yielded dissociation constants of approximate ly 3 x 10(-7) M. Integrated rate treatment of biosensor data supports a critical examination of the assumptions on which the binding models are based and suggests a need to refine such models. Nevertheless, it provides a powerful quantitative tool for assessing the Ag-Ab binding reaction.