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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(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 a hybridoma cell line that secretes polysaccharide A from Neisseria meningitidis and prepare a monoclonal antibody. The competitive ELISA method was developed to detect polysaccharide A of group ACYW135. Methods: The hybridoma cell lines were screened by classical hybridoma technique. The ascites monoclonal antibody was harvested by intraperitoneal injection in mice. The conditions of indirect ELISA were optimized and 5 batches of ACYW135 meningitis Neisserial polysaccharide vaccine group A polysaccharide content, the test results using SPSS software as a single sample t test analysis. Results: The standard curve was log-logit and the regression equation was obtained: y = -1.664 8-2.254 2x, R2 = 0.989 5, linear range 0.022 8 ~ 1.200 7μg / mL, IC50 0.165 8μg / mL, 0.022 8μg / m L, the detection of vaccine group A polysaccharide content, the detection value and vaccine labeling amount was not statistically significant (P> 0.05). Conclusion: The hybridoma cell lines were successfully screened to prepare group A polysaccharide-specific monoclonal antibodies. The established indirect competitive ELISA method has the advantages of high sensitivity, good stability and repeatability, which can be used in the development of N. meningitidis polysaccharide vaccine Of quantitative detection, can overcome the phosphorus content and rocket immunoelectrophoresis detection A group of polysaccharide shortcomings.