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.