MEASUREMENT OF ANTIBODY-ANTIGEN DISSOCIATION-CONSTANTS USING FAST CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION

The dissociation constant (K-d) of a monoclonal antibody with fluoresc ein isothiocyanate (FITC)-labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with las er-induced fluorescence detection (CE-LIF). K-d determinations were ma de by separating free FITC-insulin and its complex with the antibody i n equilibrated solutions in 6 s or less. The use of LIF detection allo wed quantification of free and bound FITC-insulin in the picomolar ran ge, as is required to measure K-d's below 1 nM. The K-d of FITC-insuli n with the antibody was determined to be 0.25 nM by Scatchard analysis . The K-d's of the antibody with unlabeled insulins from several speci es were obtained by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration data from a series of solutions co ntaining a fixed concentration of FITC-insulin and antibody and variab le concentrations of insulin to the expected curve derived from the eq uilibria and mass balance of the solutions. K-d's for the different in sulins were between 0.34 and 0.64 nM.