ABSTRACT Viral pathogenesis kills 100 million people each year. However, broadly neutralizing antibodies (Abs) pro- duced by B lymphocytes in fortunate hosts routinely eliminates the threat of the most lethal viruses. Identifying the antigenic epitopes that can induce such neutralizing Abs (nAbs) via immunization is at best protracted and fraught with technical challenges, if possible at all. We are developing a novel, microfluidic, lab-on-a-chip tech- nology to radically speed the ability to functionally assay the neutralization capability of clonal Ab produced by one human B cell. PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated mi- crofluidics ENabled Technology) is a high-throughput, single-cell resolution platform that can measure the neu- tralization capability of the Abs produced by single B lymphocytes through a direct functional assay in a droplet microfluidics format. Thus, it provides an unbiased, single-cell resolution, high-throughput, near-complete anal- ysis of the entire B cell repertoire to identify B cells that neutralize viral infection. As influenza sometimes causes one half million deaths per year and we have a battery of tools and rea- gents already developed for this pathosystem, we have chosen it as the viral model in which to test our central hypothesis that PRESCIENT will deliver a fast and low cost route for discovering neutralizing Abs (nAbs). We will take a tripartite approach with these aims: 1) to optimize the performance of the device with greater reliabil- ity, throughput and efficiency, 2) to quantitatively assess PRESCIENT's ability to recognize hybridomas that make nAbs against H1N1 influenza from progressively more rigorous mixed populations, and 3) to rapidly iden- tify nAbs against H1N1 and H3N2 influenza from EBV-immortalized human peripheral blood B cells. Phage and other display systems commonly used for nAb discovery have inherent bias and protein produc- tion hurdles due to non-mammalian post-translational modifications, thus direct utilization of B cells is ideal. Droplet microfluidic systems, where pico-liter scale water-in-oil emulsion droplets function as independent bio- reactors, can efficiently manipulate cells with unprecedented speed and precision. Droplet microfluidic systems for screening Abs produced from hybridomas that inhibit specific biochemical reactions have been described. However, systems that integrate Ab screening and viral neutralization bioassays have not yet been achieved. The major innovation is the development and utilization of the first high-throughput system for the functional discovery of human nAbs against infectious agents, a truly vertical leap for the fields of vaccinology, immuno- therapeutic design and epitope discovery. With PRESCIENT, immunologists will be able to rapidly identify from a convalescent patient's blood draw the ?needle in the haystack? paratope that generates life-saving broadly neutralizing Abs. Ultimately, the unique immunoglobulin heavy and light chain DNA rearrangements will be iso- lated from the PRESCIENT-sorted cell for monoclonal Ab production or structural vaccine epitope engineering. 1