293 transfectin

The mRNA of human IgG and IgM heavy and light chains was amplified from single B cells as described previously [26] (link), using modified primer sets for RT-PCR (Table S1). One-step RT-PCR (OneStep RT-PCR Kit, Qiagen) was performed using forward primers in the heavy and light chain leader sequence and reverse primers in the constant region of IgG, IgM, kappa, or lambda. Reported primers were used for the nested IgM and IgG PCR [26] (link) for cloning into expression vectors, RT-PCR products were used for nested IgG-PCR with modified primers (Table S1): SalI and NheI sites were added at the 5′ and 3′ ends of the H chain, and SalI and BbvCI/NotI sites were added at the 5′ and 3′ end of the kappa or lambda light chains, respectively. The PCR products were cloned into the Ptt5 mammalian expression vector [30] (licensed from the National Research Council Biotechnology Research Institute, Montreal, QC, Canada). Heavy and light chain plasmids (IgG1 format) were co-transfected into the HEK293-6E cells using 293-Transfectin (Invitrogen). Transfected 293-6E cells were allowed to secrete antibodies in serum-free F17 media supplemented with 20% TN1 for 5 days. Antibodies were purified using protein G beads (GE Healthcare).