(1053-B) High-throughput screening of secreted antibodies using a FRET-based assay in microfluidic droplets

Abstract: Antibodies have become the fastest-growing class of biopharmaceuticals used against diseases ranging from allergies and infections to cancer. The development of immunotherapies requires detailed knowledge of immune signaling, immune cell migration, and immune repertoires, and most of these details can be addressed only on a single-cell level. Here, we showcase a droplet microfluidics-assisted approach for high-throughput screening and selection of antibody-secreting cells using a FRET-based detection assay. The FRET signal was obtained via the specific binding of the secreted antibody to two fluorescently labeled probes that were co-encapsulated with individual cells. For the proof-of-concept experiment, we encapsulated antibody-producing mouse hybridoma cells MYC 1-9E10.2 (9E10) and 357-101-4 (TNF-α) into ~23 pL droplets together with two fluorescently labeled antibodies (goat anti-mouse IgG, F(ab')₂ fragment specific antibody, conjugated to Alexa Fluor 488, and goat anti-mouse IgG, Fcγ fragment specific antibody, conjugated to Alexa Fluor 647). After encapsulation, the emulsion was incubated at standard cell culture conditions (37°C, 5% CO2) for 1 hour to allow for antibody secretion. The fluorescence-activated droplet sorting (FADS) approach was applied to select droplets containing antibody-producing cells based on the detection of the FRET signal. Upon droplet sorting, we observed specific enrichment of droplets containing antibody-secreting cells.
In conclusion, we have demonstrated the sorting of hybridoma cells based on their secreted antibodies, an application inaccessible to Fluorescence Activated Cell Sorting, which requires the physical connection of analytes to the cells. Fluorescence-activated droplet sorting is an empowering tool that enables high-throughput analysis of individual cells based on the analytes they secrete.