Measurement of antibody-antigen dissociation constants using fast capillary electrophoresis with laser-induced fluorescence detection

✍ Scribed by Li Tao; Professor Robert T. Kennedy

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 574 KB
• Volume: 18
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150180121

✦ Synopsis

Abstract

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.