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Monoclonal antibody binding affinity determined by microchip‐based capillary electrophoresis
Author(s) -
Chiem Nghia H.,
Harrison D. Jed
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150191641
Subject(s) - bovine serum albumin , chemistry , capillary electrophoresis , chromatography , scatchard plot , stoichiometry , monoclonal antibody , electrophoresis , affinity electrophoresis , binding constant , serum albumin , analytical chemistry (journal) , binding site , affinity chromatography , antibody , biochemistry , enzyme , biology , organic chemistry , immunology
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 × 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.