Rapid Antibody Production and Labeling

Amplification of immunoglobulin heavy and light chain variable genes by rapid amplification of 5’ cDNA ends PCR was performed as previously described [26 (link)]. High-throughput production of recombinant antibodies was performed by TS-jPCR as previously described [26 (link)]. Briefly, cognate pairs of linear immunoglobulin heavy and light chain genes were cotransfected into HEK293 cells, and the cell culture supernatant was used for enzyme-linked immunosorbent assay (ELISA) and cell ELISA four days after transfection. For large-scale antibody production, immunoglobulin heavy and light chain genes were inserted into pET-IgG and pET-IgK vectors by target-selective homologous recombination cloning as described previously [27 (link)]. Transient transfection of the plasmids into CHO-S cells was performed with the CHOgro High Yield Expression System (Takara Bio). Recombinant mAbs were purified from the CHO cell culture medium by using MabCaptureC™ Protein A chromatography resin (ThermoFisher Scientific). The purified antibodies were labeled with either alkaline phosphatase or biotin by using Alkaline Phosphatase Labeling Kit-SH or Biotin Labeling Kit -NH2 according to the manufacturer’s instructions, respectively (Dojindo).