title: 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.
abstract: Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted 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 concentrations 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 secreted hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclonal antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FCS and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb production 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 culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 medium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10% G-FCS was more than 99%.
authors.affiliation: Department of Microbiology, Immunological Research and Application Center, School of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey. aybayc@tr-net.net.tr
authors.forename: C
authors.initials: C
authors.lastname: Aybay
authors.forename: T
authors.initials: T
authors.lastname: Imir
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001426:Bacterial Proteins; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D005069:Evaluation Studies as Topic; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000302:isolation & purification*; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D003470:Culture Media; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G