Lumanglas A L, Wang B S
Laboratory of Immunoendocrinology, American Cyanamid Co., Princeton, NJ 08543-0400.
Hybridoma. 1993 Feb;12(1):127-33. doi: 10.1089/hyb.1993.12.127.
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.
已证明小鼠腹水含有多种生长促进活性物质。在本研究中，我们检测了腹水对杂交瘤产生效率和细胞生长的影响。将SP2/0小鼠骨髓瘤细胞注入小鼠腹腔并收集腹水。用猪生长激素（pGH）免疫的小鼠淋巴细胞与骨髓瘤细胞按照标准杂交程序进行融合。然后将这些细胞接种到含有2.5%腹水或20%胎牛血清（FCS）的培养基的96孔板中，并培养数天。收集这些培养物的上清液并分析抗pGH抗体。结果表明，与补充FCS的培养基相比，补充腹水的培养基产生特异性抗体分泌杂交瘤的效率提高了4倍。此外，将杂交瘤细胞培养在微量滴定板中，发现其对腹水的增殖反应呈剂量依赖性。用胰蛋白酶预先消化腹水可消除这种效应，表明其蛋白质性质。由于抗IL-4和IL-6抗体未能改变腹水的刺激作用，因此B淋巴细胞相关细胞因子似乎不太可能参与其中。使用Superose 12柱通过快速蛋白质液相色谱（FPLC）对腹水进行分级分离，发现活性部分是一种小分子量肽（<1000道尔顿）。因此，在杂交瘤技术领域，小鼠腹水能够替代培养基中的传统FCS。
