摘要
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 develop and preliminarily apply a double antibody sandwich ELISA method for determination of polysaccharide contents and molecular size of group W135 Neisseria meningitidis（Nm）in groups A,C,Y and W135 menignococcal polysaccharide vaccine（MPV4）.Methods A double antibody sandwich ELISA method was developed using polyclonal antibody against group W135 Nm as coating antibody,of which the working concentrations of coating antibody and enzyme-labeled antibody were optimized by checkerboard titration method.The developed ELISA method was used for quantitative determination of group W135 Nm,and verified for reproducibility of linearity,specificity,accuracy,precision and quantitative limit,then used for identification of 10 batches of group W135 Nm polysaccharide and 10 batches of unrelated Nm polysaccharide.The polysaccharide content,molecular size and recovery rate of MPV4 were determined by the developed ELISA method.Results The optimal working concentrations of coating and enzyme-labeled antibodies were 10 μg / ml and 1 ∶ 15 000 respectively.The dose-response curve of the developed ELISA method showed high linearity within a concentration range of 2.5 ~ 20 ng / ml,with a correlation coefficient of more than 0.99.The determination results of group W135 Nm polysaccharide by the developed method was strong positive,while those of unrelated Nm polysaccharide were negative.The coefficients of variation of results of intra-and inter-assay on polysaccharide contents of 16,8 and 4 ng / ml group W135 Nm polysaccharide were 1.1% ~ 9.0%,while the recovery rate was 87.5% ~ 105.0%,and the quantitative limit was 4 ng / ml.Both the coincidence rates of positive results of group W135 Nm polysaccharide and negative results of unrelated Nm polysaccharide were 100%.All the determination results of polysaccharide contents,molecular sizes and recovery rates of 3 batches of MPV4 met the requirements in application for temporary regulations for group W135 Nm polysaccharide.Conclusion The developed double antibody san
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第12期1688-1691,1696,共5页
Chinese Journal of Biologicals
关键词
脑膜炎球菌多糖疫苗
W135群
多糖
分子大小
酶联免疫吸附测定
Meningococcal polysaccharide vaccine
Group W135
Polysaccharide
Molecular size
Enzyme linked immunosorbent assay
