利用DNA免疫技术制备人CLDN18.2单克隆抗体  

Preparation of specific monoclonal antibodies against human CLDN18.2 through DNA immunization

作　　者：戎卓娜 赵传科[1] 孟麟[1] 王冰[1] 钟堂武 王立新 寿成超(指导)[1] RONG Zhuona;ZHAO Chuanke;MENG Lin;WANG Bing;ZHONG Tangwu;WANG Lixin;SHOU Chengchao(Key Laboratory of Carcinogenesis and Translational Research,Department of Biochemistry and Molecular Biology,Peking University Cancer Hospital&Institute,Beijing 100142,China)

机构地区：[1]北京大学肿瘤医院暨北京市肿瘤防治研究所,生物化学与分子生物学研究室,恶性肿瘤发病机制及转化研究教育部重点实验室,北京100142

出　　处：《中国免疫学杂志》2022年第19期2378-2383,共6页Chinese Journal of Immunology

摘　　要：目的:制备CLDN18.2特异性单克隆抗体并对其特性和在免疫检测中的应用进行鉴定,为CLDN18.2的抗体药物研发奠定基础。方法:将in vivo-jetPEI-Gal和pcDNA3.1-CLDN18.2质粒在体外混合后经尾静脉免疫小鼠,筛选效价较高的小鼠进行细胞融合和单克隆筛选,制备CLDN18.2的特异性单克隆抗体,然后对获得的单克隆抗体的亲和力和特异性及其在细胞ELISA、流式细胞术、细胞免疫荧光和免疫沉淀等实验中的应用进行鉴定。结果:共获得15株有较好特异性的CLDN18.2单克隆抗体,其与CLDN18.2结合的EC50s均在纳摩尔或亚纳摩尔级,可在ELISA、免疫沉淀、流式细胞术和细胞免疫荧光实验中用于对CLDN18.2的特异检测,但均不能用于Western blot实验。结论:成功制备并获得了15株可识别蛋白天然构象、亲和力高、特异性好的小鼠抗人CLDN18.2单克隆抗体,为CLDN18.2抗体药物的研发奠定了重要基础。Objective:To prepare CLDN18.2-specific monoclonal antibody and identify its characteristics and application in immunoassay,so as to lay a foundation for the development of CLDN18.2 antibody drug.Methods:In vivo-jetPEI-Gal and pcDNA3.1-CLDN18.2 plasmids were mixed in vitro to immunize mice through the tail vein,and the mice with higher titers were selected for cell fusion and monoclonal screening to prepare CLDN18.2 specific antibody.The affinity and specificity of obtained monoclonal antibodies and their applications in experiments such as cell ELISA,flow cytometry,cell immunofluorescence and immunoprecipitation were identified.Results:Fifteen hybridoma cell lines of CLDN18.2 monoclonal antibodies with good specificity were obtained,and their EC50s for binding to CLDN18.2 were in nanomolar or sub-nanomolar levels,which could be applied in the specific recognition of CLDN18.2 in ELISA,immunoprecipitation,flow cytometry and immunofluorescence assays,but not in Western blot.Conclusion:Fifteen mouse anti-human CLDN18.2 monoclonal antibodies with high affinity and good specificity which native conformation of protein can be recognized were successfully prepared,thereby providing basis for the research and development of anti-CLDN18.2targeted treatment.

关 键 词：CLDN18.2 DNA免疫 单克隆抗体 制备 鉴定 

分 类 号：R735.9[医药卫生—肿瘤]