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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R2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 double-antibody sandwich ELISA for detection of influenza virus nucleoprotein (NP) in influenza vaccine. Methods: Recombinant NP was expressed in vitro as a reference substance for determination of protein content by ELISA. The concentration of the capture antibody, the initial concentration and the dilution of the antibody were screened. The ELISA method was established and validated. Results: The purity of recombinant NP obtained was above 99.0%. ELISA parameters established and optimized: capture antibody 4μg · m L-1, detection antibody 1: 1 000. The initial concentration of NP content reference substance is 300 ~ 600 ng · m L-1, and the coefficient of determination R2 of the four-parameter fitting S-curve is greater than 0.95, which is specific to the influenza A virus NP in the vaccine. The standard recoveries ranged from 88.2% to 95.3%. The reproducibility of the method (n = 5) was less than 15% RSD. Conclusion: The ELISA method of influenza virus NP in the established influenza vaccine has good specificity, accuracy and precision, and can be used for the detection of influenza virus NP in vaccine samples.