Investigation of human chorionic somatomammotropin--antihuman chorionic somatomammotropin antibody binding by two physicochemical methods: phase partition and fluorescence polarization
Urios, P.; Rajkowski, K.M.; Engler, R.; Cittanova, N.
Analytical Biochemistry 119(2): 253-260
ISSN/ISBN: 0003-2697 PMID: 7072947 DOI: 10.1016/0003-2697(82)90582-6
The determination of binding parameters for interactions between proteins and nondialyzable ligands is studied by 2 physicochemical methods, both rigorously respecting the binding equilibrium: 2-polymer aqueous phase partition and fluorescence polarization. These 2 methods are applied to an antigen/antibody system (human chorionic somatomammotropin/antihuman chorionic somatomammotropin Ig). A fluorescein-labeled antigen is used for both methods, permitting their comparison. The optimization of the phase partition system is described as in the mathematical treatment of the fluorescence measurements. The results obtained for the instrinsic apparent Ka and the number of sites (n) are Ka = 0.75 .times. 107 M-1, n = 0.16 by phase partition, and Ka = 0.83 .times. 107 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 (Ka = 1.2 .times. 108 M-1, n = 0.05). Given that, at similar ligand concentrations, the 2 methods yield identical results while respecting the binding equilibrium, their relative practicability is discussed.