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

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.

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.

Single-microchannel, multistep assay reports protein size and immunoaffinity.

We introduce a fully integrated multistep protein assay that reports both protein identity and size. To report these two properties, a microfluidic design strategy integrates pore limit electrophoresis (PLE) with a heterogeneous immunoassay in a single microchannel (PLE-IA). PLE-IA was applied in a study of follistatin, a 31.5 kDa glycoprotein regulating mammalian cell proliferation and differentiation. In a single-channel multistage assay approach, an antibody to follistatin was first immobilized in a polyacrylamide PLE gradient gel, near the origin of the separation axis. Immobilization relies on pore-limit exclusion of the antibody and not on chemical functionalization of either the sieving matrix or the antibody, making assay customization by an end-user straightforward. Subsequently, target and ladder protein species were electrophoretically introduced into the antibody-patterned PLE channel. Species having an affinity for the immobilized antibody were detected via heterogeneous immunoassay. Noninteracting and, thus, unbound species electromigrated past the patterned antibodies, along the separation axis, and finally separated according to the pore-size limit of each, yielding a log-linear dependence of molecular weight on migration distance. Separations of 10 min yielded an average peak capacity of 18 ± 1.3 (separation resolution (SR) = 1) in a 10 mm separation distance. Comparison of the separated peaks in two parallel PLE channels in the presence or absence of capture antibody with a protein size ladder revealed good agreement of the target molecular weight with reported values. In addition, a more than 50-fold decrease in the detection limit (0.078 vs 5 nM) was achieved using an electrophoretic "continuous injection" technique in which sample material was continuously loaded for 40 min. On the basis of this proof-of-principle demonstration with follistatin, PLE-IA should find application in study of cell signaling, including questions related to aging and regeneration.