人附睾蛋白4单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against human epididymisprotein 4, HE4

目的建立人附睾蛋白4(HE4)杂交瘤细胞株,并对其分泌的HE4单克隆抗体进行鉴定和初步应用,为HE4的功能研究和人源化抗体的制备奠定基础。方法使用pcDNA3.0-HE4重组质粒免疫BALB/C小鼠,采用PEG融合技术建立杂交瘤细胞株,制备单克隆抗体。通过免疫荧光法、有限稀释法及金标斑点法测定单克隆抗体的交叉反应性及免疫球蛋白的类型和亚类。结果获得2株可稳定分泌HE4单克隆抗体的杂交瘤细胞株,分别命名为1C5和1C8,均为IgMκ类。2株单抗能够与表达HE4蛋白的卵巢癌细胞发生反应,而不与Sp2/0细胞、CHO细胞等发生交叉反应。杂交瘤细胞株1C5培养上清效价为1∶160,腹水效价为1∶1 280,而杂交瘤细胞株1C8培养上清效价为1∶128,腹水效价为1∶1 024。结论成功地制备出两株抗HE4单克隆抗体,均具有良好的特异性,为进一步建立免疫分析方法,进行HE4相关研究奠定了良好的基础。

Objective To construct and identify the hybridoma cell lines for human epididymisprotein 4（HE4）, and lay a foundation of using HE4 monoclonal anitboies for HE4 functional study and humanized antibody preparation for early diagnosis and treatment of human ovarian cancer. Methods BALB / C mice were immunized with recombinant plasmid pcDNA3.0-HE4. Hybridoma cell lines were obtained using PEG fusion technology to prepare monoclonal antibodies. The immunoglobulin class, subclass, and cross-reaction of the mAbs were analyzed by immunofluorescence, limiting dilution and dot immunogold methods. Results Two hybridoma cell lines were obtained, which can stably secrete HE4 mAbs namely 1C5 and 1C8, and both were IgMκ class. The two mAbs reacted to the X cells that can express HE4, but did not cross-react to Sp2 / 0 cells and CHO cells. The culture supernatant titer of hybridoma cell line 1C5 was 1 ∶160, and peritoneal fluid was 1∶1 280. The culture supernatant titer of hybridoma cell line 1C8 was 1∶128 and peritoneal fluid was 1∶1 024.Conclusion Two mAbs against HE4 with high specificity were successfully established, which lay a good basis for further studies of HE4 and human ovarian cancer.