DETERMINATION OF ANTIGEN-ANTIBODY AFFINITY BY IMMUNOCAPILLARY ELECTROPHORESIS

The use of affinity capillary electrophoresis for the characterization of antigen-antibody interactions (immunocapillary electrophoresis) is shown using monoclonal antibodies against phosphotyrosine as a model system. The influence of the interaction kinetics on the peak profiles was demonstrated in experiments with addition of phosphotyrosine to t he electrophoresis buffer. One of the two antibodies that were tested exhibited peak broadening while the other showed no change in peak sha pe but had a decreased mobility proportional to the amount of phosphot yrosine present. The migration shifts which were of the order 0.05 to 0.15 min at 439 V/cm were a consequence of the antibody-antigen comple xes having a slower mobility than the non-complexed antibody. On the b asis of measurement of migration shifts at different antigen concentra tions, dissociation constants were estimated and shown to be independe nt on the applied field strength. Thus, when certain requirements are fulfilled, immuno-capillary electrophoresis is a fast and simple metho d for establishing binding characteristics of unlabelled antigen and a ntibody molecules under non-denaturing conditions and consumes minute amounts of sample.