Effect of murine ascites on the ability of hybridoma cells to produce antibody and proliferate in vitro.
Murine ascites has been shown to contain a variety of growth promoting activities. In this study, we examined the effects of ascites fluid on the efficiency of hybridoma production and cell growth. SP2/0 mouse myeloma cells were injected into peritoneal cavities of mice and ascitic fluid was collected. Lymphocytes from mice immunized with porcine growth hormone (pGH) were fused with myeloma cells in a standard hybridization procedure. These cells were then dispensed in 96-well plates in medium containing either 2.5% ascites or 20% fetal calf serum (FCS) and cultured for few days. Supernatants from these cultures were collected and analyzed for anti-pGH antibodies. It was demonstrated that ascites-supplemented medium increased the efficiency in generating specific antibody-secreting hybridomas by 4-fold over FCS-supplemented medium. Furthermore, hybridoma cells were cultured in microtiter plate and found to proliferate in response to ascites in a dose dependent manner. This effect was abolished by prior digestion of ascites with trypsin, indicating its protein nature. B-lymphocyte related cytokines seemed less likely involved because antibodies to IL-4 and IL-6 failed to alter the stimulatory effect of ascites. Ascites was fractionated by FPLC using Superose 12 column and the active moiety was found to be a small m.w. peptide (< 1,000 dalton). Therefore, murine ascites is capable of substituting for conventional FCS in culture medium in the area of hybridoma technology.
Lumanglas
A L
AL
Laboratory of Immunoendocrinology, American Cyanamid Co., Princeton, NJ 08543-0400.
Wang
B S
BS
D000818:Animals; D000917:Antibody Formation / Q000187:drug effects; D001202:Ascitic Fluid / Q000737:chemistry*; D002455:Cell Division / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D013006:Growth Hormone / Q000276:immunology; D006133:Growth Substances / Q000494:pharmacology*; D006825:Hybridomas / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D013552:Swine; D013729:Terpenes; D014407:Tumor Cells, Cultured
D006133:Growth Substances; D013729:Terpenes; C009042:pristane; D013006:Growth Hormone