A群脑膜炎奈瑟球菌多糖单克隆抗体的制备及其应用  

Preparation of group A Neisseria meningitidis polysaccharide and its applications

作　　者：舒曼[1] 鲁卫东[1] 钱雯 马波 叶红艳[1] 黄微 兰萍[1] Shu Man Lu Weidong Qian Wen Ma Bo Ye Hongyan Huang Wei Lan Ping(Key Laboratory of Natural Medicine Pharmacology,Kunming Medical University,Kunming 650500 yunnan Wosen Biotechnology Co. Ltd, Kunming 650106, China)

机构地区：[1]昆明医科大学暨云南省天然药物药理重点实验室,云南昆明650500 [2]沃森生物技术股份有限公司,云南昆明650106

出　　处：《南京医科大学学报（自然科学版）》2016年第11期1339-1343,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：云南省高新技术产业发展项目([2014]1264)

摘　　要：目的 ：筛选能分泌A群脑膜炎奈瑟球菌多糖抗体的杂交瘤细胞株,制备单克隆抗体,运用该单抗建立检测ACYW135群脑膜炎奈瑟球菌多糖疫苗中A群多糖的竞争ELISA方法。方法 ：运用经典的杂交瘤技术筛选杂交瘤细胞株,采用小鼠腹腔注射收获腹水型单克隆抗体,优化条件建立间接竞争ELISA,分别测定5支同一批和3个不同批次的ACYW135群脑膜炎奈瑟球菌多糖疫苗成品中的A群多糖含量,检测结果应用SPSS软件做单样本t检验分析。结果：标准曲线经Log-logit处理,得到回归方程：y=-1.664 8-2.254 2x,R^2=0.989 5,线性范围0.022 8~1.200 7μg/m L,IC50为0.165 8μg/m L,最低检测限为0.022 8μg/m L,检测疫苗中A群多糖含量,检测值与疫苗标示量无统计学差异（P〉0.05）。结论 ：成功筛选杂交瘤细胞株,制备A群多糖特异性单克隆抗体,建立的间接竞争ELISA方法 ,灵敏度高、稳定性好、可重复性强,可用于脑膜炎奈瑟球菌多糖疫苗研发过程中的定量检测,能克服磷含量法和火箭免疫电泳法检测A群多糖含量的缺点。Objective：To screen and obtain hybridoma cells secreting antibody of group A Neisseria meningitidis polysaccharide,produce monoclonal antibody,and establish an indirect compete ELISA based on Mc Ab to detect A polysaccharide content in group ACYW135 meningococcal polysaccharide vaccine. Methods：Hybridoma cells were screened by hybridoma technology,Mc Ab was produced by injecting cells into mice abdominal cavity,the indirect compete ELISA was developed through optimizing the test conditions,A polysaccharide content was detected in 5 groups of ACYW135 meningococcal polysaccharide vaccines from the same batch and 3 from different patches separately. Data were analyzed through one-samples T test in SPSS. Results：The equation of linear regression after Log-logit processing：y=-1.664 8-2.254 2x,R^2=0.989 5,the linear detection range was 0.022 8~1.200 7 μg/m L,IC50=0.165 8 μg/m L,and the lowest detection limit of this method was 0.022 8 μg/m L. A polysaccharide content was detected in the vaccines. There were no significant differences between detected value and original value（P〉0.05）. Conclusion：We successfully screened the hybridoma cell and produced the Mc Ab,provided indirect compete ELISA with good sensitivity,stability and repeatability,which detects A polysaccharide content in the process of meningococcal vaccine. It can overcome the difficulties that phosphorus content method and rocket eletroimmunoassay have.

关 键 词：杂交瘤细胞株 单克隆抗体 流脑疫苗 

分 类 号：R392.11[医药卫生—免疫学]