High-throughput affinity profiling of antibodies against soluble and native membrane targets by digital flow cytometry
Binding affinity measurements are essential for optimizing and validating therapeutic and research antibodies, yet no high-throughput platform supports quantitative affinity measurements for both soluble and native membrane targets. Here we present a suite of digital flow cytometry (dFC) assays that determine equilibrium binding affinities by directly counting free and bound molecules in homogeneous mixtures without immobilization. The assays enable titration-free absolute KD measurements for fluorescent binding partners, affinity measurements for unlabeled antibodies against membrane receptors on cell-derived extracellular vesicles (EVs), and multiplexed assays for rapid ranking or screening of antibody variants. We demonstrate applications by quantifying the effects of degree-of-labeling on the affinity of antibody-dye conjugates, and of drug conjugation on therapeutic antibody binding to receptors on human cancer EVs. By combining single-molecule quantitation, adaptable assay designs, and high-throughput analysis, dFC provides a versatile platform for therapeutic antibody optimization and research antibody validation.