Hitrap kappaselect

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).

Supernatants from transiently transfected CHO cells were collected, and then cleared by centrifugation at 3500 g for 10 minutes at RT. Chimeric anti-BLG antibodies were purified using either HiTrap KappaSelect (chimeric human IgE antibodies) or HiTrap protein A HP (chimeric human IgG1 antibodies) columns (GE Healthcare) on an ÄKTAprime plus liquid chromatography system (GE Healthcare). Briefly, culture supernatants were adjusted to pH 7.2 and loaded onto affinity columns. After washing with PBS pH 7.2, chimeric anti-BLG antibodies were eluted from affinity columns with 0.1 M glycine buffer pH 3.0, and instantly neutralized in 1 M Tris buffer pH 9.0. After dialysis against PBS pH 7.2, antibody concentrations were determined by measuring the absorbance at 280 nm. Extinction coefficients (1 mg/ml at 1 cm pathway) of 1.53 and 1.36 [22] were used for chimeric IgE antibodies and chimeric IgG1 antibodies, respectively. Purity of chimeric anti-BLG antibodies was analyzed with SDS-PAGE using a pre-casted 4–12% BisTris gel/MOPS running buffer NuPage Novex system (Invitrogen) followed by Coomassie Brilliant Blue staining (BioRad). MW Magic ladder (BioRad) was run in parallel to estimate the molecular weight of proteins.