抗乳腺癌多形上皮黏蛋白1单克隆抗体的制备及初步应用

Preparation and preliminary application of anti-human breast cancer MUC1 monoclonal antibody

目的 制备并鉴定抗多形上皮黏蛋白1（MUC1）单克隆抗体,将抗体应用于人乳腺癌组织、纤维腺瘤、囊性增生症及正常乳腺组织的临床鉴别,采用免疫组化方法进行检测.方法 用合成的MUC1重复序列短肽与钥孔血蓝蛋白（KLH）及牛血清白蛋白（BSA）偶联后作为抗原免疫Balb/c小鼠,杂交瘤技术筛选出稳定分泌单克隆抗体的细胞株.制备单克隆抗体,以ELISA、Western blot、流式细胞术、免疫荧光法对抗体进行特异性鉴定及亚类分析;将抗体应用于人乳腺癌组织、纤维腺瘤、囊性增生症及正常乳腺组织的临床鉴别,采用免疫组化方法进行检测.结果 获得4株稳定分泌抗MUC1的杂交瘤细胞株,第1株反应性最强,效价1∶10 7以上.抗体亚型3株为IgG1,1株为IgM,轻链均为κ型.经Western blot、流式细胞术、免疫荧光、免疫组化检测证实抗体与乳腺癌细胞株MCF-7特异性结合,单抗检测MUC1在乳腺癌组织与纤维腺瘤、囊性增生症及正常乳腺组织之间表达差异明显（均P＜0.01）.结论 抗MUC1单克隆抗体经检测效价高、特异性强,为乳腺癌早期诊断和靶向治疗的进一步研究奠定了基础.

Objective To prepare the anti-human MUC1 monoclonal antibody （mAb） and to apply it in human breast cancer tissues,fibro adenoma,cystic hyperplasia and normal breast tissues,using immunohistochemistry assay.Methods Synthetic MUC1 repetitive sequence short peptide coupled with KLH protein and BSA protein as antigen to immunize Balb/c mice,cell lines capable of secreting monoclonal antibodies were screened by hybridoma cell fusion technique.The specificities and subclasses of the prepared monoclonal antibodies were identified and analyzed by ELISA,Western blot,flow cytometry and immunofluorescence test.The antibodies were used in the detection of clinical human breast cancer,benign fibro adenoma and normal breast tissue by immunohistochemical methods.Results Four hibridoma cell lines secreting specific anti-MUC1 mAbs were obtained,The titers of the most powerful antibody were above 1 ∶ 107.3 strains were of IgG1 antibody,one was of IgM,and the light chain of which was categorized into chain to.The mAb could specifically bind to the MCF-7 cells,which was verified by Western blot,flow cytometry analysis,fluorescence immunoassay and immunohistochemistry test.Significant differences of expression （all P ＜ 0.01） of the detection of MUC1 monoclonal antibodies were found in breast cancer tissue,fibro adenoma,cystic hyperplasia and normal breast tissues.Conclusions The mAb against MUC1 developed in this study is of high titers and specific for breast cancer,exhibiting a favourable prospect for early diagnosis and targeted therapy of breast cancer.