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目的获得印记基因父系表达基因10(PEG10)单克隆抗体(mAb),为进一步研究PEG10的功能以及肝癌的实验室诊断奠定基础。方法采用细胞融合技术,以PEG10抗原免疫BALB/c小鼠的脾细胞与Sp2/0骨髓瘤细胞进行融合,通过多次阳性筛选和有限稀释,建立了抗人PEG10 mAb杂交瘤细胞株。采用间接ELISA和Southern biotech试剂盒对mAb的特性进行鉴定,采用Western blot法及免疫组织化学染色验证mAb与肝癌细胞内源性PEG10蛋白的结合。结果成功建立2株稳定分泌抗PEG10蛋白的mAb杂交瘤细胞株,分别命名为3E8和3F3,均属IgG1亚类,κ型,且无交叉反应。Western blot法及免疫组织化学实验证实,mAb 3E8均能特异性结合肝癌细胞内源性PEG10蛋白。结论成功制备了效价高、特异性好的PEG10 mAb,该抗体有可能用于肝癌的实验室诊断。
OBJECTIVE: To obtain monoclonal antibody (mAb) of parent gene 10 (PEG10) of imprinted gene and lay foundation for further study on the function of PEG10 and the laboratory diagnosis of liver cancer. Methods The spleen cells of BALB / c mice immunized with PEG10 antigen were fused with Sp2 / 0 myeloma cells by cell fusion technique. After several positive screening and limited dilution, the anti-human PEG10 mAb hybridoma cell lines were established. The characteristics of mAb were identified by indirect ELISA and Southern biotech kit. The binding of mAb to the endogenous PEG10 protein of hepatoma cells was verified by Western blot and immunohistochemistry. Results Two mAb hybridoma cell lines stably secreting anti-PEG10 protein were successfully established and named as 3E8 and 3F3, respectively, belonging to IgG1 subclass and κ type without cross-reaction. Western blot and immunohistochemistry confirmed that mAb 3E8 could specifically bind to the endogenous PEG10 protein of hepatoma cells. Conclusion The PEG10 mAb with high titer and specificity was successfully prepared and could be used for the laboratory diagnosis of liver cancer.