摘要
目的:制备特异性CD161单克隆抗体,对其生物学特性进行检测与评估,并筛选出其中具有功能的单克隆抗体。方法:构建CD161/L929过表达细胞株;以CD161-His蛋白为免疫原,采用小鼠杂交瘤融合技术获得稳定分泌抗体的杂交瘤细胞株,通过流式细胞术和ELISA筛选抗体效价较高的杂交瘤细胞株进行多次亚克隆;用双阳性杂交瘤细胞株免疫小鼠,诱导腹水产生,制备单克隆抗体并纯化;筛选可用于Western blot和免疫组化的单克隆抗体;同时筛选可阻断CD161-LLT1结合的单克隆抗体。结果:成功构建了CD161/L929过表达细胞株。共筛选出40株可用于ELISA的单克隆抗体,29株可用于流式细胞术的单克隆抗体,6株可用于Western blot的单克隆抗体,4株可用于免疫组化的单克隆抗体;获得多株抗体可阻断CD161-LLT1结合的抗体,其中13A1、13A9和10A3亲和力较高,具有竞争阻断性。结论:本研究制备出多株特异性CD161单克隆抗体,筛选出可应用于各种免疫学实验的抗体,并筛选出其中具有阻断性的抗体,为进一步探究CD161发挥作用的机制提供物质基础。
Objective:To prepare specific CD161 monoclonal antibodies,detect and evaluate their biological characteristics,and screen out functional monoclonal antibodies.Methods:Constructed CD161/L929 overexpressing cell lines;used CD161-His protein as immunogen,obtained hybridoma cells stably secreting antibodies using mouse hybridoma fusion technology,screened hybridoma cells with high antibody titers by flow cytometry and ELISA for multiple subcloning;immunized mice with double-positive hybridoma cells to induce ascites production for monoclonal antibody preparation and purification;screened monoclonal antibodies suitable for Western blot and immunohistochemistry;simultaneously screened monoclonal antibodies which could blocking CD161-LLT1 binding.Results:CD161/L929 overexpression cell line was successfully constructed.Screened out 40 monoclonal antibodies suitable for ELISA,29 monoclonal antibodies suitable for flow cytometry,6 monoclonal antibodies suitable for Western blot,4 monoclonal antibodies suitable for immunohistochemistry;several obtained antibodies could block CD161-LLT1 binding,among which 13A1,13A9 and 10A3 had high affinity and competitive blocking ability.Conclusion:This study successfully prepares multiple specific CD161 monoclonal antibodies,screenes antibodies applicable to various immunological experiments,and screenes out blocking antibodies,providing a material basis for further exploration of the mechanism of CD161 action.
作者
王昱涵
曹磊
牛亚燕
胡玉敏
吕红艳
赵华阳
吴妹英
WANG Yuhan;CAO Lei;NIU Yayan;HU Yumin;LYU Hongyan;ZHAO Huayang;WU Meiying(Suzhou Medical College of Soochow University,the Affiliated Infectious Diseases Hospital,Suzhou 215000,China;The Fifth People's Hospital of Suzhou,the Affiliated Infectious Diseases Hospital of Soochow University,Suzhou 215000,China;Jiangsu Institute of Clinical Immunology,the First Affiliated Hospital of Soochow University,Suzhou 215000,China;Suzhou Xuguang Kexing Antibody Biotechnology Co.,Ltd.,Suzhou 215000,China)
出处
《中国免疫学杂志》
北大核心
2026年第2期446-450,共5页
Chinese Journal of Immunology
基金
苏州市科技局科技“科教兴卫”项目(KJXW2022046)
苏州市呼吸系统传染性疾病临床医学中心项目(Szlcyxzx202108)。
关键词
CD161
单克隆抗体
杂交瘤技术
生物学特性
CD161
Monoclonal antibodies
Hybridoma technology
Biological characterization
