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 fluorescein isothiocyanate (FITC)-labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). K-d determinations were made by separating free FITC-insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC-insulin in the picomolar range, as is required to measure K-d's below 1 nM. The K-d of FITC-insulin 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 species were obtained by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC-insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. K-d's for the different insulins were between 0.34 and 0.64 nM.