H因子结合蛋白抗原含量双抗夹心ELISA检测方法的优化与应用

Optimization and application of DAS-ELISA for detecting fHBP antigen

目的优化双抗夹心-酶联免疫吸附法(double-antibody sandwich enzyme-linked immunosorbent assay,DAS-ELISA)检测H因子结合蛋白(factor H-binding protein,fHBP)抗原含量的方法,并将其应用于重组B群脑膜炎球菌疫苗体外相对效力评价。方法筛选4种抗fHBP单克隆抗体(monoclonal antibodies,McAb)适宜的抗体组合,通过方阵滴定法确定适宜的捕获抗体和酶标检测抗体稀释倍数、包被液、包被条件和显色时间,对优化后方法的专属性、线性、检测限、精密度和准确度进行了验证,并利用该方法检测fHBP蛋白相关制品的抗原含量。结果确定了适宜的双抗体检测组合以及试验条件(以柠檬酸缓冲液,4℃孵育16 h),在以柠檬酸缓冲液包被捕获抗体5.0000μg/mL、检测抗体1∶2000稀释、显色时间15 min的条件下,A450 nm-fHBP浓度曲线相关系数(r)>0.98,平行孔CV<10%,检测限<0.0002μg/mL,精密度<15%,准确度(直接回收率)<20%。亚家族之间抗原检测无干扰。多批fHBP相关制品检测结果稳定,差异无统计学意义(P>0.05)。结论优化后的方法可用于fHBP抗原含量的检定。

Objectivee To optimize the method of double-antibody sandwich enzyme-linked immunosorbent assay(DASELISA)for detecting factor H-binding protein(fHBP)antigen.This method was applied to evaluate the relative potency of recombinant meningococcal group B vaccine in vitro.Methods A matched antibody pair for ELISA was screened from four kinds of anti-fHBP monoclonal antibodies(McAb).Optimization of the concentrations of capture antibody and detection antibody were determined by square array titration,and the specificity,linearity,limit of detection,precision and accuracy of the method were validated.The method was used to detect the antigen of fHBP-based vaccine.Results The matched antibody pair and optimal conditions for ELISA were determined.The working concentration of capture antibody was 5.0000μg/mL(incubate for 16 h at 4℃).A good concentration for detection antibody was a dilution of 1:2000.The incubation time for TMB substrate was 15 min.The correlation coefficient of the curve was>0.98.The coefficient of variation for this method were<10%and the limit of detection was<0.0002μg/mL.The precision and accuracy of this method were<15%and<20%,respectively.No interference in antigen detection between subfamilies.The results of fHBP antigen from different batches were stable.Conclusion This DAS-ELISA method could detect fHBP antigen.