qRT-PCR快速检测汉滩病毒抗体中和活性实验方法的建立

摘要

目的:建立一种基于qRT-PCR的微量细胞中和实验改良法,快速、定量检测汉滩病毒感染中的中和抗体活性.方法:将本室前期制备的抗汉滩病毒高中和活性鼠源性单抗3G1、鼠/人嵌合单抗1D9和感染剂量为100 TCID50的病毒混合液于37℃孵育1h后感染Vero-E6细胞,提取细胞总RNA.根据GenBank数据库中汉滩病毒76-118株S基因序列设计特异性引物,在上述实验基础上以细胞总RNA为检测样品,建立qRT-PCR快速、定量检测汉滩病毒感染中中和抗体活性的实验方法.结果和结论:建立了一种基于qRT-PCR的微量细胞中和实验改良法,可定量测定汉滩病毒感染中的中和抗体活性,该法具有快速、灵敏、特异和重复性好等优点.%Objective:To develop a fast and accurate evaluation system for neutralization antibodies against Hantaan virus.Methods:Vero-E6 cells were incubated at 37℃ for 1 hour in the presence of Hantaan virus at 100 TCID50 infectious dose and candidate antibody(mAb 3G1 or murine/human chimeric mAb 1D9).The total cellular RNAs from the incubation mix were extracted.The specific primers were designed according to the S gene sequence of Hantavirus 76-118 in the GenBank database.The extracted total RNAs as template,qRT-PCR was performed to detect the effect of neutralization antibodies for Hantaan virus.Results & Conclusion:The method called qRT-PCR-based micro-cell-neutralization test was successfully established,which is capable to quantify the neutralizing antibodies rapidly and acurrately.