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以北京鸭红血球为抗原免疫Balb/cJ小鼠,三天后取脾细胞与小鼠NS-1骨髓瘤细胞在PEG作用下,用两种方法进行细胞融合。只有淋巴细胞杂交瘤细胞能在一种选择性培液——HAT液中生长,具有分泌抗体活性的杂交瘤细胞,借助其上清液的斑点试验加以选择。对斑点试验阳性的杂交瘤细胞进行克隆化和扩大培养,得到能专一分泌抗北京鸭红血球单克隆抗体的淋巴细胞杂交瘤,定名为CBH-1。在体外经过69代培养,历时十月余,分泌能力仍未见衰减。体内可传4—5代,腹水中可检出大量分泌抗体。用降植烷、Arlacel A和石蜡油诱发动物的腹水,其量以降植烷最高、石蜡油次之。使用国产石蜡油作为诱发腹水试验似具有一定实用价值。加5γ转铁蛋白至每毫升不含牛血清的DMEM液中,在15天的培养期内,结果至少与加20%小牛血清的培液的结果相似。首次克隆化后的CBH-1细胞中,空斑形成细胞达97.5%,再次克隆化的细胞中,空斑形成细胞达99.8%,证明是均一的细胞群落。本文还就杂交瘤制备过程及细胞稳定性问题进行了讨论。
Balb / cJ mice were immunized with Beijing duck erythrocytes as antigen. Three days later, spleen cells and mouse NS-1 myeloma cells were fused by two methods under the action of PEG. Only lymphocyte hybridoma cells can grow in a selective culture medium, HAT solution, with hybridoma cells secreting antibody activity, selected by the spot test of the supernatant. Splotch-positive hybridoma cells were cloned and expanded to obtain a monoclonal antibody specific for the secretion of anti-Pekin duck erythrocyte lymphocyte hybridoma, named CBH-1. In vitro after 69 generations of culture, which lasted more than 10 months, the secretion capacity has not seen decay. Can pass 4-5 generations in vivo, ascites can detect a large number of secreted antibodies. Animal ascites was induced with pristane, Arlacel A, and paraffin oil in amounts that gave the highest amount of phytan and the paraffin oil followed. Use of domestic paraffin oil as induced ascites test seems to have some practical value. Add 5γ transferrin to each ml of DMEM without bovine serum, and the result is at least similar to that of 20% fetal bovine serum in a 15-day culture. In the first cloned CBH-1 cells, plaque-forming cells reached 97.5%, and again cloned cells, plaque-forming cells reached 99.8%, proved to be a homogeneous cell population. This article also discussed the hybridoma preparation process and cell stability issues.