测定脑膜炎球菌疫苗C群多糖的双抗体夹心ELISA法的建立

目的 建立测定A、C、Y、W135群脑膜炎球菌多糖疫苗(groups A,C,Y,W135 meningococcal polysaccharide vaccine,MPV4)C群多糖含量的双抗体夹心ELISA法.方法 制备抗C群多糖多克隆抗体,所得的多克隆抗体经辛酸-硫酸铵沉淀法纯化后,用过碘酸钠法对其标记辣根过氧化物酶.分别将抗C群多糖多克隆抗体作为包被抗体和酶标二抗,建立双抗体夹心ELISA法,优化反应条件,对C群多糖进行特异性定量测定.结果 建立的双抗体夹心ELISA法特异性较好,未检出与A、Y、W135群多糖的交叉反应.C群多糖在2.5～20.0 ng/ml质量浓度范围的剂量反应曲线线性最佳,相关系数大于0.99.该法的准确度和精密度均较好,试验内和试验间变异系数和多糖回收率分别为0.6％ ～9.1％和87.5％ ～ 100.0％,定量限度为4.0 ng/ml.采用该法测定3批MPV4显示,C群多糖含量及多糖分子大小KD值和回收率的测定结果均与先前的测定结果一致,均符合暂行质量标准.结论 建立的双抗体夹心ELISA法可用于MPV4 C群多糖的关键质量指标测定.

Establishment of a double antibody sandwich ELISA for determination of group C polysaccharide in meningococcal polysaccharide vaccine

 Objective  To establish a double antibody sandwich ELISA for determination of group C polysaccharide in groups A,C,Y,W135 meningococcal polysaccharide vaccine (MPV4).   Methods  The prepared polyclonal antibodies against group C polysaccharide were purified by octanoic acid-ammonium sulfate precipitation method, and then labeled with horseradish peroxidase by sodium periodate method. The polyclonal antibodies against group C polysaccharide were used as coated antibodies and secondary antibodies to establish a double antibody sandwich ELISA. The reaction conditions of the established ELISA were optimized, and specific quantitative determination of group C polysaccharide were carried out by the established ELISA.  Results  The specificity of the double antibody sandwich ELISA for determination of group C polysaccharide was high, and no cross reaction with groups A, Y and W135 were detected. The best linearity in dose-response curve of group C polysaccharide was found in a range of 2.5-20.0 ng/ml (r＞0.99). The precision and accuracy of the established ELISA were good, coefficients of variation and recovery rates of intra- and inter-assay were 0.6%-9.1% and 87.5%-100.0%, respectively. quantitation limit was identified as 4.0 ng/ml. The results of group C polysaccharide contents, molecular dimension KD value and recovery rates of three batches of MPV4 detected by the established ELISA were in accordance with those of previous determination and temporary quality control standards.  Conclusion   The double antibody sandwich ELISA can be applied to detecting the key quality indexes of group C polysaccharide in MPV4.