Production and Purification of Recombinant Antibodies

As described previously (7 (link)), human embryonic kidney fibroblast 293T cells (HEK-293T, ATCC CRL 11268^TM) were split into culture dishes (100 × 20 mm) in high glucose DMEM (HyClone, GE Healthcare), supplemented with heat inactivated fetal bovine serum (FBS, 10%), glutamine (1%) and penicillin-streptomycin (1%, HyClone, GE Healthcare). After 16–24 h, at 70–80% confluence, the medium was replaced with SFM4Transfx-293 (HyClone, GE Healthcare) supplemented with 10% FBS, 1% glutamine and 1% penicillin-streptomycin (HyClone, GE Healthcare). Cells were co-transfected 8 h later, using Lipofectamine LTX Reagent (Invitrogen), with IgH and IgL encoding plasmids (5 μg). Supernatants were collected after 7 days, and the antibodies were purified on protein G-sepharose beads (GE Healthcare, Life Technologies). Glycine buffer (0.1 M, pH 3.5) and Tris-HCl (1 M, pH 8) were used for antibody elution and pH neutralization, respectively. Purified antibody was dialyzed in PBS and its concentration was determined by Nanodrop. Antibody integrity was assessed on NuPAGE 4–12% BisTris gels (Invitrogen) stained with Coomassie blue.