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.

Bioanalytical interaction studies executed by preincubation affinity capillary electrophoresis

“…Chiem and Harrison [114] used pre-eq CZE in a microchip format for investigation of fluorescently labeled BSA-mAb interactions. Recently the continuous frontal analysis method was transferred to the microchip format.…”

“…However, in other studies LIF detection has most often been applied in order to reach the detection limits required for characterization of high affinity antigenantibody systems, e.g. [60,[114][115][116]. Traditional immunoassays do not allow distinction between binary and tertiary complexes.…”

Recent advances in the study of biomolecular interactions by capillary electrophoresis

“…With such advantages as: (i) easy adaptation, (ii) integration of sampling, separation, detection, and other unit processes, and (iii) fast separation. Chiem et al [48] used chip CE to investigate the interaction of bovine serum albumin (BSA) with anti-BSA in , 60 s. The changes in peak height for fluorescently labeled BSA were due to the binding. Two affinity complexes were separated, one identified by Scatchard analysis with a binding stoichiometry of 1:1, and the other as anti-BSA 2 -BSA complex.…”

Characterization of the interaction between human complement protein C4 and a single‐chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

“…Most applications of CE in the field of protein interactions deal with small, charged molecules as ligands [14,15]. The use of ACE for studying protein-protein interactions is limited to a few examples of high-affinity systems, especially antigen-antibody interactions [16][17][18]. The main problem in the field of moderate-to-low affinity protein-protein interactions (K D = 10 28 -10 24 M) using ACE is the high protein concentration present in the separation buffer for the mobility-shift assay.…”