In the reporting period we have completed a study demonstrating the influence of surface immobilization strategy, as well as the choice of sensor surface, on the resulting distribution of binding affinity and kinetic rate constants for a soluble binding partner. Using a monoclonal antibody against β2-microglublin as a model system, we studied binding to surfaces with different length of carboxymethyl dextran as a polymeric immobilization matrix, functionalized to different densities, and applying either random amine coupling or streptavidin capture. We found surface with short polymer coating to provide the most uniform distribution of sites that are best accessible for the analyte diffusion. This study also demonstrated the utility of our previously developed affinity distribution method in the optimization of sensor surfaces.
|Zhao, Huaying; Gorshkova, Inna I; Fu, Gregory L et al. (2013) A comparison of binding surfaces for SPR biosensing using an antibody-antigen system and affinity distribution analysis. Methods 59:328-35|
|Zhao, Huaying; Brown, Patrick H; Schuck, Peter (2011) On the distribution of protein refractive index increments. Biophys J 100:2309-17|
|Schuck, Peter; Zhao, Huaying (2010) The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing. Methods Mol Biol 627:15-54|
|Gondeau, Claire; Corradin, Giampietro; Heitz, Frédéric et al. (2009) The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer. Mol Biochem Parasitol 165:153-61|