Production and Purification of IgG1 Antibodies

For production of purified full-length IgG1 antibodies, the V_HDJ_H and V_L genes were cloned into the pCDNA3.1⁺ (Invitrogen) mammalian expression vector containing either the human IgG1 constant region gene, the human lambda light-chain constant region, or the lambda light-chain constant region gene using standard recombinant DNA technology (45 (link)). For the production of purified nCoV617Fab antibody, a stop codon TGA was introduced after the sequence (5′-TCTTGTGACAAA-3′) encoding amino acid residues, SCDK, just before the hinge of the human IgG1 constant region (46 (link)). Recombinant IgG1 antibodies and the nCoV617Fab antibody were produced in 293F cells cultured in serum-free medium by cotransfection with the generated full-length IgG1 or Fab heavy- and light-chain gene expression plasmid pairs using polyethylenimine (47 (link)). Full-length IgG1 antibodies were purified by using protein A column chromatography as described previously (45 (link)). The nCoV617Fab antibody used for the crystal structure was purified by Lambda FabSelect, an affinity resin designed for the purification of human Fab with a lambda light chain (GE Healthcare) (46 (link)).