Using affinity capillary electrophoresis to evaluate average binding constant of 18-mer diphospho-tyrosine peptide to antiphosphotyrosine Fab

We used affinity electrophoresis in capillaries to investigate the interact ion between a monovalent antiphosphotyrosine antibody fragment, antigen-bin ding fragment (Fab), and a divalent antigen (dAg), an 18-mer diphosphopepti de phosphorylated on two-site tyrosine residues. The migration shift behavi or of Fab in electrophoretic solution was observed and the quantitative exp ression was presented to estimate the arithmetical average value of the int rinsic affinities for two epitopes on the dAg with the Ag binding site on t he Fab. In dAg excess, based on measurement of mobility changes of Fab anal ytes at different dAg concentrations, the experimental average dissociation constant (<(K-d)over bar> = 27.7 mu m) was calculated, it was also found t hat the structural variation of the two epitopes for binding specificity to the Ag-binding domain of Fab is not apparent. Moreover, the <(K-d)over bar > values of Fab-dAg complexes were measured at higher electric fields and s hown to be independent of changes in the electric field. Thus, under condit ions where the total dAg concentration is in excess of the total Fab concen tration, the method and quantitative expression which we developed is gener ally useful for the understanding of molecular interaction for an unlabeled monovalent receptor and its divalent ligand in free solution.