Studies on stabilization of a human-mouse heterohybridoma producing sperm immobilizing antibody by recombinant DNA technology.

为了稳定人-鼠杂交瘤(H6-3C4)精子固定化单抗(IgM，lambda)的编码基因，从杂交瘤中克隆了重排的人免疫球蛋白mU链基因。克隆的重链(VH)基因V区与人免疫球蛋白Gamma1-重链恒定区(C-Gamma 1)基因连接。通过原生质体融合和电穿孔分别将重链基因和克隆的lambda链基因导入小鼠骨髓瘤细胞系X63Ag8.653，获得了5个稳定的转化子。培养上清液经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法鉴定，转印到硝酸纤维素膜上。然后用过氧化物酶标记的抗人伽马链或波长链抗体进行染色。在非还原条件下，所有转化子均分泌出相对分子质量为160Kd的完整形式的人免疫球蛋白G分子。在还原条件下，观察到人γ链的54Kd带和人Lambda链的25Kd带。转化子培养上清液的精子固定抗体效价为10~20单位，分泌的单抗为0.2~0.5微克/毫升，H6-3C4杂交瘤细胞培养上清液中的SI50效价约为5,000单位，IgM浓度约为2.0微克/毫升。如果考虑到IgG1的补体依赖精子固定活性低于IgM，重组抗体的亲和力似乎与原始H6-3C4单抗相同。因此，我们成功地建立了稳定生产人IgG1的转化子，该转化子完全保留了原始精子固定人单抗IgM的特异性。

For stabilizing the gene coding for sperm immobilizing monoclonal antibody (IgM, lambda from a human-mouse hetero-hybridoma (H6-3C4), the human rearranged immunoglobulin mu-chain genes were cloned from the hybridoma. The cloned V region of the heavy chain (VH) gene was ligated to human immunoglibulin gamma1-heavy chain constant region (C gamma 1) genes. This resulted in the heavy-chain class-switched from mu-chain to gamma 1-chain of H6-3C4 asntibody.The class-switched heavy-chain gene as well as the cloned lambda-chain gene were introduced into mouse myeloma cell line X63Ag8.653 by protoplast fusion and electroporation respectively.Five stable transformants were established. The culture supernatants were electrophoresed on a SDS-PAGE and the proteins were transblotted to the nitrocellulose membranes. Followed by staining with peroxidase conjugated anti-human gamma-chain or lambda-chain antibodies. Under non-reducing condition, all transformants secreted the complete form of human IgG molecules with a molecular weight of 160Kd. Under reduciht condition, the 54Kd band of human gamma-chain and 25Kd band of human lambda-chain were observed. The titers of sperm immobilizing antibody of the culture supernatants of the transformants were in the range of 10-20 units in SI50 titer.The amounts of monoclonal antibodies secreted from the transformants were about 0.2-0.5umg/ml. The SI50 titers and IgM concentrations in the supernatant of H6-3C4 hybridoma were about 5,000 units and 2.0mug/ml, respectively. If we considered the lower complement-dependent sperm immobilizing activity of IgG1 as compared to IgM, the affinity of the recombinant antibody appeared to be the same as that of the original H6-3C4 monoclonal antibody. Thus we succeeded in establishing the stable transformants producing human IgG1 which fully retained the specificity of the original sperm immobilizng human monoclonal IgM.