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 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%.
Aybay
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C
Department of Microbiology, Immunological Research and Application Center, School of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey. aybayc@tr-net.net.tr
Imir
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T
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
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D003470:Culture Media; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G