四价脑膜炎球菌多糖疫苗中结合物多糖抗原含量双抗体夹心ELISA检测方法的建立及验证

Development and verification of double antibody sandwich ELISA for quantitative determination of conjugates in quadrivalent meningococcal polysaccharide vaccine

目的建立一种检测A、C、W135和Y群四价脑膜炎球菌多糖疫苗中各血清群多糖蛋白结合物的双抗体夹心定量ELISA检测方法,并进行验证。方法以白喉类毒素无毒变异体（CRM197）特异性单抗为包被抗体,HRP标记的脑膜炎球菌A、C、W135和Y群多糖特异性单抗为捕获抗体,建立可定量检测脑膜炎球菌各血清群结合物多糖含量的双抗体夹心ELISA法,确定方法的最佳线性范围和检测限,并对方法的特异性、准确性和重复性进行验证。结果定量检测A群脑膜炎球菌结合物（GAMP-CRM197）多糖抗原含量的双抗体夹心ELISA法的线性范围为3.75~120 ng/ml,检测限为3.75 ng/ml;定量检测C群脑膜炎球菌结合物（GCMP-CRM197）多糖抗原含量的双抗体夹心ELISA法的线性范围为1.875~30 ng/ml,检测限为0.938 ng/ml;定量检测W135群脑膜炎球菌结合物（GWMPCRM197）多糖抗原含量的双抗体夹心ELISA法的线性范围为7.5~240 ng/ml,检测限为7.5 ng/ml;定量检测Y群脑膜炎球菌结合物（GYMP-CRM197）多糖抗原含量的双抗体夹心ELISA法的线性范围为1.875~60 ng/ml,检测限为0.938 ng/ml。A、C、W135、Y群双抗体夹心ELISA法均能特异性地检测相应的结合物多糖抗原含量,而不与其他群的结合物多糖抗原发生交叉反应;A、C、W135、Y群双抗体夹心ELISA法的回收率均在80%~120%之间,检测相应的结合抗原样品时,变异系数（CV）均小于15%。结论建立的双抗体夹心ELISA法特异性较强,准确性较高,重复性良好,可用于四价脑膜炎球菌多糖疫苗中各血清群结合物多糖抗原的定量检测。

Objective To develop and verify a double antibody sandwich ELISA for quantitative determination of the con-jugates in groups A, C, W135 and Y meningococcal polysaccharide vaccine. Methods A double antibody sandwich ELISA was developed using monoclonal antibody against CRM197, an avirulent mutant of diphtheria toxoid, as coating antibody, and HRP-labeled monoclonal antibody against groups A, C, W135 and Y meningococcal polysaccharide as cap-ture antibody, then determined for optimal linear range and limit of detection（LOD）, and verified for specificity, accuracy and reproducibility. Results The linear range and LOD of the developed double antibody sandwich ELISA were 3. 75 ~120 ng / ml and 3. 75 ng / ml for group A meningococcal polysaccharide（GAMP）-CRM197 conjugate, 1. 875 ~ 30 ng / ml and 0. 938 ng / ml for group C meningococcal polysaccharide（GCMP）-CRM197, 7. 5 ~ 240 ng / ml and 7. 5 ng / ml for group W135 meningococcal polysaccharide（GWMP）-CRM197, and 1. 875 ~ 60 ng / ml and 0. 938 ng / ml for group Y meningococcal polysaccharide（GYMP）-CRM197, respectively. The developed method was suitable for determination of polysaccharide antigen contents in the corresponding conjugates, which showed no cross reactions with the polysaccharide antigens of other groups. The recovery rates of polysaccharide antigens of four groups were 80% ~ 120%, whil e the coefficients of variation（CVs）of the corresponding polysaccharide antigens were less than 15%. Conclusion The developed double antibody sandwich ELISA showed high specificity, accuracy and reproducibility, which might be used for the quantitative determination of polysaccharide antigens in quadrivalent meningococcal polysaccharide vaccine.