抗HCCR单克隆抗体的制备及其鉴定

Preparation and identification of monoclonal antibody against human cervical cancer oncogene

目的制备抗人宫颈癌癌基因(HCCR)单克隆抗体(mAb),并进行鉴定。方法以HCCR重组蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞SP2/0进行细胞融合,常规筛选杂交瘤细胞。腹水诱导法制备抗人HCCR单克隆抗体,并经蛋白A亲合层析进行纯化。用Western blot和免疫荧光等方法检测HCCR mAb的特异性。用纯化HCCR蛋白进行包被,建立间接ELISA方法,检测系列稀释的HCCR mAb,确定其灵敏度。结果筛选出2株分泌抗人HCCR mAb的杂交瘤细胞株。Western blot结果表明抗人HCCR抗体能与HCCR蛋白特异性结合,免疫荧光结果显示肝癌HepG2细胞中胞浆和胞膜均呈阳性染色。间接ELISA方法检测HCCR mAb的灵敏度可达到1μg/L。结论成功制备并鉴定了特异性抗人HCCR mAb的杂交瘤细胞株,为进一步深入研究HCCR的生物学特征和临床应用打下基础。

Objective To prepare and identify monoclonal antibody against human cervical cancer oncogence（HCCR）.Methods Purified recombinant HCCR protein was used as antigen to immunize BALB/c mice.The isolated spleen cells from the immunized mice were fused with SP2/0.Monoclonal antibodies against HCCR were prepared by normal hybridoma technology.Purified mAb was prepared by protein A chromatography.To identify the characteristics of HCCR mAbs,Western blot and immunofluorescence method were employed.The indirect ELISA was established with the purified recombinant HCCR protein,and used to detect the sensitivity of mAbs against HCCR.Results Two hybridoma cell lines which could secrete anti-human mAb against HCCR were established by hybridoma technique.Specificity of mAb against HCCR was assessed by Western blot.Immunofluorescence showed that HCCR positive staining occurred in the cell hyalomitome and membrane of hepatic carcinoma HepG2.The indirect ELISA was established with the purified HCCR protein,and with this indirect ELISA,as low as 1μg/L of functional anti-human mAb against HCCR could be detected.Conclusion Human HCCR hybridoma cell lines and their secreted mAbs have been successfully obtained and identified,which lays a solid foundation to further study the biological characteristics and clinical applications of HCCR.