Investigation of human chorionic somatomammotropin-antihuman chorionic somatomammotropin antibody binding by two physicochemical methods: Phase partition and fluorescence polarization

Abstract The determination of binding parameters for interactions between proteins and nondialyzable ligands is studied by two physicochemical methods, both rigorously respecting the binding equilibrium: two-polymer aqueous phase partition and fluorescence polarization. These two methods are applied to an antigen/antibody system (human chorionic somatomammotropin/antihuman chorionic somatomammotropin immunoglobulins). A fluorescein-labeled antigen is used for both methods, permitting their comparison. The optimization of the phase partition system is described as is the mathematical treatment of the fluorescence measurements. The results obtained for the intrinsic apparent association constant ( k a ) and the number of sites ( n ) are k a = 0.75 × 10 7 m −1 , n = 0.16 by phase partition, and k a = 0.83 × 10 7 m −1 , n = 0.15 by fluorescence polarization. At low ligand concentrations (10 −8 m) fluorescence polarization measurements permitted detection of an antibody population of higher affinity ( k a = 1.2 × 10 8 m −1 , n = 0.05). Given that, at similar ligand concentrations, the two methods yield identical results while respecting the binding equilibrium, their relative practicability is discussed.