DETERMINATION OF THE ASSOCIATION CONSTANT FOR SLOW PHOSPHOSERINE-ANTI-PHOSPHOSERINE INTERACTION KINETICS BY CAPILLARY-ZONE-ELECTROPHORESIS

Capillary zone electrophoresis (CZE) was used to study the interaction of a monoclonal anti-phosphoserine antibody with its antigen. A model system that allows the determination of the real binding constant in a solution based on the change in peak areas at different concentratio ns of phosphoserine has been used for studying slow monoclonal antibod y-antigen interaction kinetics involving low-molecular-weight antigens . CZE was applied in preincubation experiments. The slow interaction k inetics led to band broadening and resulted in far lower efficiency of the separation of complexed antibody from unbound antibody. However, when the run-to-run reproducibility of free phosphoserine was examined , it was found that it can be recovered quantitatively under electroph oresis conditions. On the basis of measurement of peak areas at differ ent phosphoserine concentrations, the association constant was estimat ed (K-a = 5.21 X 10(5) M-1) and shown to be in close agreement with th at obtained by equilibrium dialysis (K-a = 4.65 X 10(5) M-1). As long as the antigen participating in the interaction can be detected and re covered quantitatively in the CZE system, the method is generally usef ul for the study of monoclonal antibody-antigen interaction where the kinetics is slow and where the charge/mass ratio of the unbound antige n differs from that of the complexed molecule.