14型肺炎球菌多糖双抗体夹心ELISA检测方法的建立

Establishment of double-antibody sandwich ELISA for detection of pneumococcal capsular polysaccharide serotype 14

目的建立并验证测定14型肺炎球菌多糖(pneumococcal capsular polysaccharide serotype 14,PS14)浓度的双抗体夹心ELISA。方法采用Protein G亲和层析纯化兔血清,得到兔抗PS14多克隆抗体(多抗)。以鼠抗PS14单克隆抗体(单抗)为捕获抗体,兔抗PS14多抗为检测抗体,碱性磷酸酶标记山羊抗兔IgG为二抗,PS14为参考品建立双抗体夹心ELISA。确定鼠抗PS14单抗和兔抗PS14多抗的工作浓度,建立标准曲线,并验证该方法的准确性、精密度和特异性,考察佐剂对该方法的影响。结果鼠抗PS14单抗的最适工作浓度为5 μg/ml,兔抗PS14多抗的最适工作浓度为1 μg/ml,标准曲线的范围为9.375 ~ 600.000 ng/ml,线性良好,相关系数为0.999。测定多糖样品的PS14回收率为110% ~ 140%,多糖蛋白结合物样品的PS14回收率为90% ~110%。平均批内和批间精密度分别是5.64%和7.14%。13价结合物混合样品的PS14回收率为85%~100%;含有佐剂的疫苗样品的PS14回收率为85% ~110%。结论成功建立了双抗体夹心ELISA,该方法准确性、精密度、特异性良好,且检测结果不受佐剂的影响,具有一定耐用性。

Objective To establish and validate a double-antibody sandwich-ELISA for detection of pneumococcal capsular polysaccharide serotype 14(PS14)concentration.Methods Rabbit anti-PS14 polyclonal antibody(pAb)was purified from rabbit serum by Protein G affinity chromatography.Double antibody sandwich-ELISA was established using mouse anti-PS14 monoclonal antibody(mAb)as capture antibody,rabbit anti-PS14 pAb as detection antibody,alkaline phosphatase-labeled goat anti-rabbit IgG as secondary antibody,and PS14 as reference.The working concentrations of mouse anti-PS14 mAb and rabbit anti-PS14 pAb were optimized to set up the standard curve.The accuracy,precision,and specificity of the method were verified.Influence of adjuvant on the method was observed.Results The optimal working concentration of mouse anti-PS14 mAb was 5μg/ml,and that of rabbit anti-PS14 pAb was 1μg/ml.The standard curve ranged from 9.375 to 600.000 ng/ml with good linearity,and the coefficient of determination was 0.999.The recovery rate of polysaccharide samples was 110%~140%,and that of polysaccharide-protein conjugate samples was 90%~110%.The mean intra-and inter-assay coefficients of variation were 5.64%and 7.14%,respectively.The recovery rate of PS14 in 13-valent pneumococcal conjugated mixture sample was 85%~100%.The recovery rate of PS14 in the vaccine sample containing adjuvant was 85%~110%.Conclusion The double antibody sandwich ELISA was successfully established.The method has good accuracy,precision,and specificity.The detection results are not affected by adjuvant,showing certain durability of the method.