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目的建立检测A、C、Y、W135群脑膜炎球菌(Neisseria meningitidis,Nm)多糖疫苗(Groups A,C,Y,W135 menig-nococcal polysaccharide vaccine,MPV4)中W135群多糖含量和分子大小的双抗体夹心ELISA法,并进行初步应用。方法以抗W135群Nm多克隆抗体作为包被抗体,建立双抗体夹心ELISA法,采用棋盘滴定法筛选包被抗体与酶标抗体的最佳工作浓度,对W135群Nm多糖进行特异性定量测定,并验证线性关系的重复性;对建立的ELISA法进行特异性、准确度、精密度及定量限的验证;采用建立的ELISA法检测10批W135群Nm多糖样品和10批无关流脑多糖样品,进行W135群Nm多糖的鉴别试验;采用建立的ELISA法测定MPV4多糖含量、多糖分子大小和回收率。结果经棋盘滴定法确定双抗体夹心ELISA法的最佳包被抗体工作浓度为10μg/ml,最佳酶标抗体工作浓度为1∶15 000稀释,W135群Nm多糖在2.5~20 ng/ml浓度范围内剂量反应曲线线性关系良好,相关系数大于0.99。采用建立的双抗体夹心ELISA法检测W135群Nm多糖为强阳性,检测其余样品的结果均为阴性;试验内及试验间测定16、8、4 ng/ml W135群Nm多糖含量的变异系数在1.1%~9.0%之间,回收率在87.5%~105.0%之间,定量限为4 ng/ml;检测W135群Nm多糖的阳性符合率和无关多糖的阴性符合率均为100%。采用该法测定3批MPV4中W135群多糖含量、分子大小及回收率均符合申报MPV4疫苗暂行规程关于W135群Nm多糖的质量标准。结论建立的双抗体夹心ELISA法可用于MPV4中W135群多糖含量和分子大小的测定。
OBJECTIVE To establish a double antibody assay for the polysaccharide content and molecular size of W135 polysaccharides in Groups A, C, Y, W135 Neisseria meningitidis (Nm) polysaccharide vaccine (Groups A, C, Y, W135 menig- nococcal polysaccharide vaccine Sandwich ELISA method, and the initial application. Methods Anti-W135 monoclonal antibody was used as the coating antibody to establish a sandwich ELISA. The optimum concentration of coating antibody and enzyme-labeled antibody was screened by checkerboard titration. The specificity of Nm polysaccharide in W135 group was determined. And the repeatability of the linear relationship was verified. The specificity, accuracy, precision and limit of quantification of the established ELISA were validated. The ELISA assay was used to detect 10 batches of Nm polysaccharide samples of W135 group and 10 batches of unrelated cerebrospinal fluid samples, W135 group of Nm polysaccharide identification test; using established ELISA assay MPV4 polysaccharide content, polysaccharide molecular size and recovery. Results The optimum antibody concentration of sandwich ELISA was 10μg / ml. The optimum concentration of ELISA enzyme was 1:15 000. The concentration of Nm polysaccharide in W135 group was 2.5-20 ng / ml The dose response curve within the range of good linear relationship, the correlation coefficient greater than 0.99. The double antibody sandwich ELISA was used to detect the Nm polysaccharide of W135 group was strongly positive. The results of the other samples were negative. The coefficient of variation (CV) of Nm polysaccharide content in W135 group The recoveries ranged from 87.5% to 105.0%, and the limit of quantification was 4 ng / ml. The positive coincidence rates of Nm polysaccharides in W135 group and those with unrelated polysaccharides were all 100%. The method was used to determine the polysaccharide content, molecular size and recovery of W135 polysaccharide in three batches of MPV4, all in compliance with the quality standard of W135 group Nm polysaccharide declared by the Provisional Regulations of the MPV4 Vaccine. Conclusion The established sandwich ELISA can be used to determine the polysaccharide content and molecular size of W135 in MPV4.