检测脑膜炎球菌多糖疫苗Y群多糖含量和分子大小的双抗体夹心ELISA法的建立和初步应用

目的建立测定A、C、Y、W135群脑膜炎球菌多糖疫苗（groupsA，C，Y，W135meningococ—calpolysaccharidevaccine，MPV4）Y群多糖含量的双抗体夹心ELISA法。方法采用杂交瘤技术制备抗Y群多糖单克隆抗体，并通过过碘酸钠法用辣根过氧化物酶标记单克隆抗体。分别以抗Y群多糖不同位点的单克隆抗体作为包被抗体和酶标二抗，通过优化反应条件来建立双抗体夹心ELISA法，同时进行方法学验证。结果建立的双抗体夹心ELISA法特异性良好，未检出与A、C、W135群多糖的交叉反应。Y群多糖在2．5～20．0ng／ml范围的剂量反应曲线呈现最佳线性关系，相关系数〉0．99。该法的试验内和试验间准确度较好，精密度较佳，定量限度为4ng／ml。采用该法测定3批MPV4Y群多糖显示，Y群多糖的含量、多糖分子大小‰值和回收率的结果均与先前的检定结果一致，符合暂行质量标准。结论建立的双抗体夹心ELISA法可用于MPV4Y群多糖的关键质量指标测定。

Establishment and preliminary application of a double antibody sandwich ELISA for determination of contents and molecular dimension of group Y polysaccharide in meningococcal polysaccharide vaccine

Objective To establish a double antibody sandwich ELISA for determination of group Y polysaccharide contents in groups A, C, Y, W135 meningococcal polysaccharide vaccine （MPV4）. Methods The monoclonal antibodies against group Y polysaccharide were prepared by hybridoma technique and labeled with horseradish peroxidase by sodium periodate method. The monoclonal antibodies against different sites of group Y polysaccharide were used as coated antibodies and secondary antibodies, respectively. The reaction conditions of the double antibody sandwich ELISA were optimized. The established ELISA was validated by a series of tests. Results The established ELISA had better specificity, and no cross reactions with groups A, C and W135 were detected. The best linear correlation in dose-response curve of group Y polysaccharide was found in a range of 2.5-20.0 ng/ml, and correlation coefficient values were all 〉 0.99. The precisions and accuracies of intra- and inter-assay for the established ELISA were both good, and the quantitation limit was 4 ng/ml. The results of contents, molecular dimension KDvalue and recovery rates of group Y polysaccharide in three batches of MPV4 detected by the established ELISA were in accordance with those of previous determination and temporary quality control standards. Conclusion The established double antibody sandwich ELISA can be applied to determining the key quality indexes of group Y polysaccharide in MPV4.