Ohnishi K, Chiba J, Goto Y, Tokunaga T
J Immunol Methods. 1987 Jun 26;100(1-2):181-9. doi: 10.1016/0022-1759(87)90188-8.
In order to define the optimum conditions of electrofusion technique for the generation of antibody-producing hybridomas, mouse spleen cells or EBV-transformed human B cells were fused with mouse myeloma cells (SP2/0) or human fusion partner cells (KR-4 or KR-12), respectively, by electric field pulse under various conditions. The results confirm reports that the presence of both Ca2+ and Mg2+ in fusion medium and pretreatment of mixed cells with proteases improve hybridoma yield. Moreover, the presence of liposome or hydrophobic protein in the fusion medium greatly enhanced the yield. Under optimum conditions, hybridoma yields of mouse cells and human cells were 2.5 X 10(-4) and 1 X 10(-4), respectively. These efficiencies were about ten times higher than those obtained by the conventional polyethylene glycol technique. Microscopic observation of the fusion-process revealed that in a human cell system 20%-50% of the cells were physically fused, although only one in 5000 physically fused human cells grew as a hybridoma after hypoxanthine-aminopterin-thymidine selection.
为了确定用于产生抗体分泌杂交瘤的电融合技术的最佳条件，分别在各种条件下通过电场脉冲将小鼠脾细胞或EB病毒转化的人B细胞与小鼠骨髓瘤细胞（SP2/0）或人融合伴侣细胞（KR-4或KR-12）进行融合。结果证实了如下报道：融合培养基中Ca2+和Mg2+的存在以及用蛋白酶对混合细胞进行预处理可提高杂交瘤产量。此外，融合培养基中脂质体或疏水蛋白的存在极大地提高了产量。在最佳条件下，小鼠细胞和人细胞的杂交瘤产量分别为2.5×10(-4)和1×10(-4)。这些效率比通过传统聚乙二醇技术获得的效率高约十倍。融合过程的显微镜观察显示，在人细胞系统中，20%-50%的细胞发生了物理融合，尽管在次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷选择后，每5000个发生物理融合的人细胞中只有一个作为杂交瘤生长。
