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We present a simple microfluidic fluorescence bead assay for accurately measuring antibody--antigen binding kinetics with a standard inverted fluorescent microscope. We measured association and dissociation rate constants from antibody--antigen interactions spanning nearly 4 orders of magnitude in equilibrium binding affinity (30 pM-100 nM). Two versions of this assay are presented, which allow for dissociation rate measurements either directly, by use of fluorescently labeled antigen, or indirectly, by use of unlabeled antigen. We also demonstrate simultaneous, multiplexed binding measurements of multiple antibody-antigen interactions using a combination of spectral separation and spatial localization. Complete antibody-antigen binding kinetics were measured for as little as 8 x [10.sup.4] antibody molecules (~132 zeptomoles) immobilized on a single bead and less than 2 x [10.sup.6] antibodies (~3 attomoles) loaded into the microfluidic device, a reduction in detection limit and sample consumption of 4 orders of magnitude when compared to surface plasmon resonance (SPR) spectroscopy and alternative measurement techniques. We show that the microfluidic bead assay, when combined with small volume compartmentalization, enables direct measurement of antigen binding kinetics of antibodies secreted from single hybridoma cells. We anticipate that this assay will be useful as a routine analytical tool for studying molecular interactions as well as for screening primary antibody-secreting plasma cells isolated from immunized animals. 10.1021/ac101956e

Here we report a low-pressure bead packing technique for the robust integration of high-performance chromatography columns in poly(dimethylsiloxane) microfluidic devices made by multilayer soft lithography (MSL). A novel column geometry featuring micrometer-sized bypass channels along the entire length of the separation channel is used to achieve rapid packing of multiple high-quality bead bed columns in parallel with near-perfect yield. Pulse tests show that these microfluidic columns achieve exceptional reproducibility and efficiency, with measured plate counts of 1 650 000/m ± 7%, corresponding to a reduced plate height of h = 0.12 ± 7%. The combination of high-performance chromatography columns and valve-based microfluidics offers new opportunities for the integration of sample processing with preparative and analytical separations for biology and chemistry. [ABSTRACT FROM AUTHOR]