Generating fast-on rate reagents for lateral flow assays to detect HCV - hepatitis c virus (HCV) is a small, enveloped RNA virus that causes hepatitis c and some forms of liver cancer and lymphoma

Many infected individuals are asymptomatic, which makes early diagnosis challenging. In the proposed work, we will apply phage-display to produce avidity- boosted and fast on-rate recombinant affinity reagents for use in capture and detection of HCV core AG in lateral flow assays (lfas). This project is a collaboration between tango biosciences, and the university of houston laboratory of prof. Richard willson. Tango is a start-up specializ- ING in innovative techniques in the phage-display and avidity engineering of combinatorial PEP- tides, antibody fragments, and antibody surrogates. DR. Willson has extensive technical and translational experience with lfas.for specific aim #1, tango will generate recombinant affinity reagents by phage-display that bind the HCV core antigen (CAG) with high affinity and fast on- rates. Tango will discover monobodies and human single-chain variable fragments (scfvs) that bind different epitopes of CAG via megaprimer shuffled tandem affinity reagents (megastar). Tango will mutagenize the coding regions of individual monobodies/scfvs and select for fast on- rates (critical in LFA) and slow off-rates. Tandem high-avidity forms of the best monobodies/ scfvs will be constructed with different length linkers for phage-display and conversion into LFA reporter nanoparticles. We will aim for the following performance metrics for optimized affinity reagents: on-rates of 108 m-1 SEC-1 and dissociation constants of = 1 PM. In specific aim #2, viri- ONS and soluble forms of the optimized affinity reagents will be formatted by DR. Willson's team as capture and detector reagents in phage lfas. After systematically testing buffers, conjugate and blocking chemistries, the goal will be to reach a limit of detection (LOD) of CAG (spiked into human plasma) of 0.5 PM (10 PG/ML), which is sensitive enough to detect 85% of HCV infec- tions. We will validate the optimized LFA format by spiking CAG over a range of concentrations into commercial, DE-identified human plasma from both sexes, a range of ages, and varied DIS- ease and metabolic backgrounds, to detect any cofounding biological variables. Finally, the best affinity reagents and prototype LFA will be evaluated in the laboratory of an HCV expert. SUC- cessful completion of this work will lead to a prototype LFA for HCV CAG and establish a RE- search paradigm applicable for the development of superior lfas for detecting infections. -1