Kinetic Rate Determination via Electrophoresis along a Varying Cross-Section Microchannel.

Abstract

High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody-antigen association and dissociation rate constants, kon and koff, in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The kon is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify koff, immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine kon and koff for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines kon and koff in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.