Modulating theKinetics of a Fluorescence AnisotropyImmunoassay Using Tracer Point Mutations to Measure Human C‑PeptideSecretion On-Chip

Fluorescence anisotropy immunoassays (FAIAs) are widely used to quantify the concentration of target proteins based on competitive binding to a monoclonal antibody with a tracer. We recently designed an FAIA to measure mouse C-peptide secretion from living islets in a continuous-flow microfluidic device (InsC-chip). To develop a similar assay for human C-peptide, we selected two monoclonal antibodies (Ab1 and Ab2) that initially showed a low dynamic range and slow kinetics. One option to measure this assay on-chip was to extend the length of the mixing channels. However, this strategy would increase dispersion and ultimately lower the temporal resolution of secreted C-peptide. To shorten the time-to-reach equilibrium for Ab1, we reengineered the tracer based on a comparison between the human and mouse C-peptide sequences, resulting in >30-fold shorter time-to-reach equilibrium. To increase the relatively small dynamic range for Ab2, we used partial epitope mapping and targeted point mutations to increase the dynamic range by 45%. Finally, we validated both FAIAs by measuring depolarization-induced secretion from individual human stem cell-derived islets in our InsC-chip. These data demonstrate a strategy to optimize FAIA kinetics to be measured in continuous-flow microfluidic devices.