Binding kinetic rates measured via electrophoretic band crossing in a pseudohomogeneous format

With relevance spanning from immunohistochemistry to immunoassays and therapeutics, antibody reagents play critical roles in the life sciences, clinical chemistry, and clinical medicine. Nevertheless, nonspecific interactions and performance reproducibility remain problematic. Consequently, scalable and efficient analytical tools for informed selection of reliable antibody reagents would have wide impact. Therefore, we introduce a kinetic polyacrylamide gel electrophoresis (KPAGE) microfluidic assay that directly measures antibody-antigen association and dissociation rate constants, kon and koff. To study antibody-antigen association, an antigen zone is electrophoresed through a zone of immobilized antibody. Upon crossing, the interaction yields a zone of immobilized immunocomplex. To quantify kon, we assess immunocomplex formation for a range of antigen-antibody interaction times. Here, interaction time is controlled by the velocity of the electromigrating antigen zone, which is determined by the strength of the applied electric field. All species are fluorescently labeled. To quantify koff, an immobilized zone of immunocomplex is subjected to in situ buffer dilution, while measuring the decay in immunocomplex concentration. Two approaches for antibody immobilization are detailed: (i) size-exclusion-based antibody immobilization via a molecular weight cutoff (MWCO) filter fabricated using polyacrylamide gel and (ii) covalent antibody immobilization realized using a photoactive benzophenone methacrylamide polyacrylamide gel. We determine kon and koff for prostate-specific antigen (PSA) and compare to gold-standard values. The KPAGE assay completes in 90 min, requiring 45 ng of often-limited antibody material, thus offering a quantitative antibody screening platform relevant to important but difficult to characterize interaction kinetics.