A群脑膜炎奈瑟球菌多糖单克隆抗体的制备及其应用

发布时间：2018-02-07 13:34

本文关键词： 杂交瘤细胞株单克隆抗体流脑疫苗　出处：《南京医科大学学报(自然科学版)》2016年11期 　论文类型：期刊论文

【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,R2=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(P0.05)。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。
[Abstract]:Objective: to screen hybridoma cell lines that secrete antibodies against Neisseria meningitidis polysaccharide in group A and to prepare monoclonal antibodies. The monoclonal antibody was used to establish a competitive ELISA method for the detection of group A polysaccharides from Neisseria meningitidis polysaccharide vaccine in ACYW135 group. Methods: hybridoma cell lines were screened by classical hybridoma technique, and ascites monoclonal antibodies were harvested by intraperitoneal injection of mice. The optimized conditions were established to establish indirect competitive ELISA.The concentration of group A polysaccharides in the finished products of five ACYW135 group meningitis Neisseria meningitidis polysaccharides vaccine was determined respectively. Results: the standard curve was processed by Log-logit, and the regression equation was obtained: the regression equation was: Yi-1.664 8-2.2542xR2N 0.989 5. The linear range was 0.022 8 渭 g / m -1. 200 7 渭 g / L IC50 was 0.165 8 渭 g / mL, the lowest detection limit was 0.022 8 渭 g / mL, and the detection limit was 0.022 8 渭 g / mL. Conclusion: the hybridoma cell line was screened successfully, the specific monoclonal antibody against group A polysaccharides was prepared, and the indirect competitive ELISA method was established with high sensitivity, good stability and strong reproducibility. It can be used for quantitative detection of Neisseria meningitidis polysaccharide vaccine, and can overcome the shortcomings of phosphorus content and rocket immunoelectrophoresis.
【作者单位】：昆明医科大学暨云南省天然药物药理重点实验室;沃森生物技术股份有限公司;
【基金】：云南省高新技术产业发展项目([2014]1264)
【分类号】：R392

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