呼吸道合胞病毒融合前F蛋白三聚体定量检测方法的建立及应用  

Establishment and application of an ELISA for the quantitation of respiratory syncytial virus prefusion F protein trimer

作　　者：唐悦 王婉 侯风萍 寇桂英 陈玥如 郭岚 李奇蒙 傅生芳 李雄雄 TANG Yue;WANG Wan;HOU Fengping;KOU Guiying;CHEN Yueru;GUO Lan;LI Qimeng;FU Shengfang;LI Xiongxiong(Lanzhou Institute of Biological Products Co.,Ltd.,Gansu Provincial Vaccine Technology Innovation Center,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司、甘肃省疫苗技术创新中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第4期16-22,共7页Progress In Microbiology and Immunology

摘　　要：目的建立呼吸道合胞病毒(respiratory syncytial virus,RSV)融合前F蛋白(prefusion F protein,pre-F)三聚体定量检测方法,并对其进行方法学验证和初步应用。方法选取RSV pre-F三聚体特异性抗体AM14作为捕获抗体,辣根过氧化物酶(horseradish peroxidase,HRP)标记的RSV pre-F特异性抗体D25作为检测抗体,通过方阵滴定法确定捕获抗体和检测抗体的最佳浓度,对封闭液进行优化,建立双抗体夹心ELISA。分别对该方法的专属性、准确度、精密度和耐用性进行验证,并应用于RSV亚单位疫苗工艺开发中pre-F三聚体的定量检测。结果该方法确定的捕获抗体AM14包被浓度为1.25μg/mL,检测抗体D25浓度为0.50μg/mL,封闭液为1%牛血清白蛋白(bovine serum albumin,BSA)。方法验证结果显示,线性范围为1.50~24.00 ng/mL,标准曲线R2>0.99,最低检测限可达1.33 ng/mL;该方法专属性良好,仅与RSV pre-F三聚体反应;准确度和精密度验证结果显示回收率为87.41%~117.03%,变异系数(coefficient of variation,CV)为5.47%~14.07%;检测抗体孵育时间及显色时间在一定范围内波动时,回收率在87.65%~106.45%,证明该方法耐用性优良;该方法可应用于RSV pre-F发酵和纯化等样品的检测。结论成功建立并验证RSV pre-F三聚体定量检测方法,该方法专属性强,准确度、精密度、耐用性均优良,可应用于RSV疫苗工艺开发中pre-F三聚体的定量检测,无需Octet或者Biacore等精密仪器,具有操作简单、定量准确、成本低、便于推广应用等优点。Objective To establish a method for quantitative detection of prefusion F protein(pre-F)trimer of respiratory syncytial virus(RSV),and to validate the method and preliminarily apply it.Methods RSV pre-F trimer-specific antibody AM14 was selected as the capture antibody,and RSV pre-F specific antibody D25 labeled by horseradish peroxidase(HRP)was used as the detection antibody.The optimal concentration of capture antibody and detection antibody was determined by square matrix titration,and the blocking solution was optimized to establish a double-antibody sandwich ELISA.The specificity,accuracy,precision and robustness of this method were verified respectively,and this method was applied to the quantitative detection of pre-F trimer in the process development of RSV subunit vaccine.Results The results showed that the coating concentration of capture antibody AM14 was 1.25μg/mL,the concentration of detection antibody D25 was 0.50μg/mL,and the blocking solution was 1%bovine serum albumin(BSA).The results of validation showed that the linear range of this method was 1.50-24.00 ng/mL,the R~2 value for the standard curve was>0.99,and the detection limit was 1.33 ng/mL.This method had good specificity,high accuracy,precision and robustness.The recovery rate was 87.41%-117.03%,and the coefficient of variation(CV)was 5.47%-14.07%.This method was used for quantitative detection of RSV pre-F trimer in fermented and purified samples.Conclusion The quantitative detection method for RSV pre-F trimer was established and verified,which showed good specificity,high accuracy,precision and robustness.This method does not require precision instruments such as Octet or Biacore and could be used for quantitative detection of pre-F trimer in the process development of RSV vaccine.The advantages of this method are accurate,low-cost and easy-operation.

关 键 词：呼吸道合胞病毒 融合前F蛋白 定量 酶联免疫吸附试验 三聚体构象 

分 类 号：R373.14[医药卫生—病原生物学]