Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells

Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapi d procedure using protein G affinity chromatography. 20 ml of FCS was deple ted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap protein G Sepharose column followed by appropriate elution. Various concen trations of IgG-depleted FCS (G-FCS) were used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G (anti-HBs IgG1 mAb producing hybridoma cell) and CAy-M (anti-HBs IgM mAb producing hybridoma cell), which secrete d hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclona l antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FC S and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb p roduction and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMI/G -FCS were similar to culture in RPMI/FCS and significantly better than cult ure in Hybri Max. We found that G-FCS was superior to whole FCS as a cultur e supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the s upernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 m edium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the pur ity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10 % G-FCS was more than 99%. (C) 2000 Elsevier Science B.V. All rights reserv ed.