Measurement of antibody-antigen dissociation constants using fast capillary electrophoresis with laser-induced fluorescence detection.
The dissociation constant (Kd) 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). Kd 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 Kd's below 1 nM. The Kd of FITC-insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The Kd'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. Kd's for the different insulins were between 0.34 and 0.64 nM.
Tao
L
L
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Kennedy
R T
RT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000378:metabolism; D006736:Horses; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism*; D007834:Lasers; D012756:Sheep; D013552:Swine; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007328:Insulin; C031573:insulin, fluorescein-isothiocyanated-; D016650:Fluorescein-5-isothiocyanate