A quantitative assessment of heterogeneity for surface-immobilized proteins

Many biotechnological applications use protein receptors immobilized on sol id supports. Although, in solution, these receptors display homogeneous bin ding affinities and association/dissociation kinetics for their complementa ry ligand, they often display heterogeneous binding characteristics after i mmobilization. In this study, a fluorescence-based fiber-optic biosensor wa s used to quantify the heterogeneity associated with the binding of a solub le analyte, fluorescently labeled trinitrobenzene, to surface-immmoblized m onoclonal anti-TNT antibodies. The antibodies were immobilized on silica fi ber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody bin ding affinity and kinetic rate parameters for these different immobilizatio n schemes. In addition, we globally analyzed kinetic data with a two-compar tment transport-kinetic model to analyze the heterogeneity in the analyte-a ntibody kinetics. These analyses provide a quantitative tool by which to ev aluate the relative homogeneity of different antibody preparations. Our res ults demonstrate that the more homogeneous protein preparations exhibit mor e uniform affinities and kinetic constants.