Recombinant Production of ITS01 Antibody

Production of recombinant ITS01 was performed as previously described (12 (link)). Briefly, HEK293T cells in 175 mm plates were transfected with 60 μg total DNA/plate at 80% confluency with PEIpro transfection reagent (Polyplus). Cells were co-transfected with the AAV ITS01 transfer plasmid and a plasmid encoding furin at a 4:1 ratio. At 16 hrs post-transfection, 10% FBS-DMEM media was replaced with serum-free 293 Freestyle media (Invitrogen). Media was collected after 48 hrs, debris was cleared by centrifugation for 10 min at 1,500 g and filtered using 0.45 μm filter flasks (Millipore Sigma). Proteins were isolated with HiTrap columns (Cytiva) and eluted with IgG Elution Buffer (Thermo Scientific) into 1M Tris-HCl Buffer, pH 9.0 (G Biosciences, St. Louis, MO). Buffer was exchanged with PBS and protein concentrated to 1-2 mg/mL with Amicon Ultra Centrifugation Filters (Millipore Sigma). Heavy and light chains of the antibodies were assessed by Coomassie-stained SDS-PAGE. Antibodies were stored at 4°C before use and frozen at -80C for long term storage.