title: Monoclonal antibody binding affinity determined by microchip-based capillary electrophoresis.
abstract: The affinity constant of a monoclonal antibody to fluorescently labeled bovine serum albumin (BSA) was measured in diluted mouse ascites fluid using a microfluidic chip to perform affinity capillary electrophoresis. Borofloat glass-based devices could be used repeatedly with samples for many months. On-chip separations were performed in less than 60 s, and 30-60 s was required for manual sample exchange. The change in peak height for BSA with increasing BSA/anti-BSA concentration ratio was used to determine concentration changes in bound and free BSA. A Scatchard plot analysis gave an affinity constant (more exactly the intrinsic association constant) of 3.5+/-0.6 x 10(7) M(-1) for a 1:1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1:1 stoichiometric ratio. The other complex is proposed to have a stoichiometry with an excess of anti-BSA to BSA, most likely (anti-BSA)2-BSA, on the basis of a faster migration time than the 1:1 complex, a decrease in the amount of this complex with increasing [BSA], and predictions of theoretical models for multi-valent antigens. Potential applications of microchip-based devices in affinity measurements are discussed.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Canada.
authors.forename: N H
authors.initials: NH
authors.lastname: Chiem
authors.forename: D J
authors.initials: DJ
authors.lastname: Harrison
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000936:Antigen-Antibody Complex / Q000032:analysis*; D001666:Binding Sites, Antibody; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000295:instrumentation; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex