甲型流感病毒NP含量ELISA检测方法的建立

Establishment of an enzyme-linked immunoassay(ELISA) method for the quantitation of influenza A NP in influenza vaccine

目的：建立一种双抗体夹心ELISA检测方法,以检测流感疫苗中甲型流感病毒的核蛋白（NP）含量。方法：体外重组表达NP作为ELISA法测定蛋白含量参考品,筛选捕获抗体和检测抗体工作浓度、起始浓度和系列稀释倍数,建立ELISA法并对其进行优化和验证。结果：获得的重组NP纯度为99.0%以上。建立和优化后的ELISA参数：捕获抗体4μg·m L^-1,检测抗体1∶1 000。NP含量参考品起始浓度为300~600 ng·m L^-1,四参数拟合S型曲线的决定系数R^2大于0.95,对疫苗中的甲型流感病毒NP具有特异性,原液和成品的加标回收率为88.2%~95.3%,方法重复性（n=5）的RSD小于15%。结论：建立的流感疫苗中甲型流感病毒NP含量ELISA检测方法特异性好,准确性和精密性高,可用于疫苗样品中甲型流感病毒NP含量的检测。

Objective： To establish a sandwich enzyme-linked immunosorbent assay（ ELISA） for quantitation of influenza A virus nucleprotein（ NP） in influenza vaccine. Methods： In vitro recombinant expressed NP was used as the ELISA protein reference for NP content determination. The ELISA method was established through the optimization of several conditions,including the working concentration of capture antibody and detection antibody,initial concentration and series dilution. Results： The purity of recombinant NP was more than 99%. The optimized parameters of ELISA method were 4 μg·m L^-1for capture antibody,1 ∶ 1 000 for detection antibody,and 300-600ng·m L^-1for initial concentration of NP reference. Determination coefficient of four- parameter fitting S curve（ R^2） was greater than 0. 95. The established ELISA method had specificity for influenza A virus NP in influenza vaccine,with more than 88. 2%-95. 3% recovery rate of the bulk and finished products,and less than 15% repeatability RSD（ n = 5）. Conclusion： The established ELISA method shows good specificity,high accuracy and high precision,and can be applied for the quantitation of influenza A virus NP in the vaccine.