Monoclonal antibody binding affinity determined by microchip-based capillary electrophoresis

Area of Science:

• Analytical Chemistry
• Biochemistry
• Microfluidics

Background:

• Accurate measurement of antibody-antigen affinity is crucial for diagnostics and therapeutics.
• Traditional methods can be time-consuming and require large sample volumes.
• Microfluidic devices offer potential for rapid, high-throughput affinity analysis.

Purpose of the Study:

• To measure the affinity constant of a monoclonal antibody to bovine serum albumin (BSA).
• To evaluate the utility of microfluidic affinity capillary electrophoresis for this measurement.
• To characterize the stoichiometry of antibody-BSA complexes.

Main Methods:

• Affinity capillary electrophoresis performed on a microfluidic chip.
• Use of fluorescently labeled BSA and mouse ascites fluid.
• Scatchard plot analysis to determine affinity constants and stoichiometry.
• On-chip separation times under 60 seconds.

Main Results:

• An affinity constant (intrinsic association constant) of 3.5±0.6 x 10^7 M^-1 was determined for a 1:1 stoichiometric ratio.
• Two distinct antibody-BSA affinity complexes were separated and characterized.
• One complex exhibited 1:1 stoichiometry, while the other likely involved a (anti-BSA)2-BSA complex.

Conclusions:

• Microfluidic affinity capillary electrophoresis is a rapid and reusable method for determining antibody-antigen affinity constants.
• The technique successfully identified and characterized multiple binding stoichiometries.
• This platform shows promise for various applications in bioanalytical measurements and drug development.