50k mwco amicon ultra centrifugal filter units

Wells containing reactive hybridomas were cloned by single-cell fluorescence-activated cell sorting. These hybridoma clones were expanded in Medium E serially into 48-well plates, 12-well plates, and T-75 cm² flasks, respectively. Hybridoma clones were expanded further into T-225 cm² flasks or G-Rex^® devices (Wilson Wolf) in serum-free medium (Hybridoma SFM [Gibco]). Supernatants were harvested after approximately 21 days, or in sets of 3 to 5 days, respectively, through a 0.2-μm pore size filter. Antibodies were purified from the filtrate using HiTrap Protein G (GE Healthcare Life Sciences), HiTrap MabSelect SuRe (GE Healthcare Life Sciences), HiTrap KappaSelect (GE Healthcare Life Sciences), HiTrap LambdaFabSelect (GE Healthcare Life Sciences), or CaptureSelect^™ IgA affinity matrix (Thermo Fisher Scientific) columns on an ÄKTA pure 25M chromatography system. Antibodies were concentrated using 50K MWCO Amicon® Ultra centrifugal filter units (Millipore) followed by desalting and buffer exchange with 7K MWCO Zeba desalting columns (Thermo Fisher Scientific).

Human mAbs were generated as previously described (Williamson et al., 2020 ). Briefly, cryopreserved PBMCs were thawed, transformed with Epstein-Barr virus (EBV), and fused with the HMMA 2.5 myeloma cell line to generate hybridomas. Hybridomas were cloned by single-cell fluorescence-activated cell sorting and mAbs were purified from hybridoma supernatant filtrate using HiTrap Protein G (Cytiva Life Sciences) or HiTrap MabSelect SuRe (Cytiva Life Sciences) columns on an ÄKTA Pure 25M chromatography system. Antibodies were concentrated using 50K MWCO Amicon® Ultra centrifugal filter units (Millipore) followed by desalting and buffer exchange with 7K MWCO Zeba desalting columns (Thermo Fisher Scientific). The humanized WNV E16 mAb has been described previously (Oliphant et al., 2005 (link)) and was purified by protein A affinity chromatography.