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目的建立发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)糖蛋白抗原(Gn)的定量双抗体夹心ELISA检测方法,并进行验证,以用于疫苗生产过程中SFTSV糖蛋白抗原含量的监测。方法用SFTSV糖蛋白抗原分别免疫新西兰兔及BALB/c小鼠,制备抗SFTSV多克隆抗体和单克隆抗体。以抗SFTSV多克隆抗体作为包被抗体,经辣根过氧化物酶标记的单克隆抗体作为酶标抗体,建立SFTSV糖蛋白抗原(Gn)定量双抗体夹心ELISA检测方法,确定该方法的线性范围、定量限,并对该方法的特异性、精密度、准确性、稳定性、适用性进行验证。结果建立的ELISA方法的线性范围为0.125~4.000μg/ml,定量限为0.125μg/ml,线性相关系数R2的平均值为0.994 1;该方法可特异性检测SFTSV病毒株,而与布尼亚病毒属其他病毒、Vero细胞培养上清及其他生产辅料均无交叉反应;该方法检测不同浓度SFTSV样品的变异系数小于15%,回收率在85%~115%之间;该检测试剂于37℃放置3 d对样品进行检测,变异系数小于15%;该方法检测SFTSV原液制备过程中不同阶段样品,随着工艺过程的不断推进,样品中单位蛋白的抗原含量呈上升趋势,可有效反映抗原纯化过程。结论建立了SFTSV糖蛋白抗原(Gn)的定量检测方法,具有较高的广谱性、灵敏度、特异性和稳定性,为疫苗生产工艺过程的质量控制奠定了基础。
Objective To establish a sandwich ELISA for detection of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) glycoprotein antigen (Gn) and to validate it for use in the production of SFTSV sugar Protein antigen content monitoring. Methods New Zealand rabbits and BALB / c mice were immunized with SFTSV glycoprotein antigens respectively to prepare anti-SFTSV polyclonal antibody and monoclonal antibody. Anti-SFTSV polyclonal antibody was used as coating antibody and horseradish peroxidase-labeled monoclonal antibody as enzyme-labeled antibody to establish a sandwich ELISA assay for detecting SFTSV glycoprotein antigen (Gn) quantitatively. The linear range of the method was determined , Limit of quantitation, and to verify the specificity, precision, accuracy, stability and applicability of this method. Results The linear range of ELISA was 0.125 ~ 4.000μg / ml, the limit of quantification was 0.125μg / ml, the linear correlation coefficient R2 was 0.994 1. This method can detect the SFTSV virus strains specifically, The virus belongs to other virus, Vero cell culture supernatant and other production materials have no cross-reaction; The method for detecting different concentrations of SFTSV sample coefficient of variation is less than 15%, the recovery rate was between 85% to 115%; The detection reagent at 37 ℃ The sample was tested for 3 days and the coefficient of variation (CV) was less than 15%. The method was used to detect the samples in different stages during SFTSV stock solution preparation. As the process progressed, the antigen content per unit protein in the sample showed an upward trend, process. Conclusion The quantitative detection method of SFTSV glycoprotein antigen (Gn) has been established. It has a high spectrum, sensitivity, specificity and stability and lays the foundation for the quality control of vaccine production process.