Isolation and Purification of Human mAbs

Human mAbs were isolated previously from healthy adults who were naturally infected with a GII.4 Sydney virus in 2013 using an established human B cell hybridoma technique (27 (link)). Here, cloned hybridoma cell lines that had been cryopreserved were thawed and placed into culture and expanded gradually from 48-well plates to 12-well plates, T-25, T-75, and eventually to multiple T-225 flasks for each cell line. Following 4 weeks of incubation at 37°C, supernatant from the T-225 flasks was harvested and filtered through a 0.4-µm filter. The supernatant was purified using column chromatography, specifically HiTrap KappaSelect and Lambda FabSelect affinity resins (GE Healthcare Life Sciences).