发热伴血小板减少综合征布尼亚病毒糖蛋白抗原(Gn)定量检测方法的建立及验证

Development and verification of auantitative detection assay for glycoprotein(Gn)antigen of severe fever with thrombocytopenia syndrome bunyavirus

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 develop and verify a double antibody sandwich ELISA for quantitative detection of glycoprotein（Gn）antigen of severe fever with thrombocytopenia syndrome bunyavirus（SFTSV）and use for monitoring of virus antigen content during SFTSV vaccine production. Methods Polyclonal and monoclonal antibodies against SFTSV were prepared from the serum of rabbits and the ascites of BALB/c mice immunized with SFTSV Gn antigen respectively. A double antibody sandwich ELISA method was developed using polyclonal antibody as coating antibody and HRP-labeled monoclonal antibody as enzyme-labeled antibody, and verified for specificity, precision, accuracy, stability and suitability.Results The linear range of the developed ELISA method was 0. 125 - 4. 000 μg/ml, with a R^2 value of 0. 994 1, while the quantitative detection limit was 0. 125 μg/ml. SFTSV strain was detected specifically by the developed method, while no cross reactions with other viruses belonging to Bunyavirus, culture supernatant of Vero cells or other subsidiary materials were observed. The recovery rates of samples at various concentrations were 85% - 115%, with CVs of less than15%. The CVs of test results by the ELISA reagent after storage at 37 ℃ for 3 d was less than 15%. The antigen content of unit protein in samples at various stages of preparation of bulk of SFTSV showed an increasing tendency, which reflected the purification process of antigen. Conclusion A quantitative detection assay for SFTSV Gn antigen was developed, which was broad spectrum, sensitive, specific and stable. It laid a foundation of quality control of production process of vaccine.