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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1 h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 establish a qRT-PCR-based microtiter cell neutralization assay for rapid and quantitative detection of neutralizing antibody activity in Hantaan virus infection. Methods: The anti-Hantaan virus high and active murine monoclonal antibody 3G1, mouse / human chimeric monoclonal antibody 1D9 and the virus dose of 100 TCID50 prepared in this study were incubated at 37 ℃ for 1 h and then infected with Vero- E6 cells, total cellular RNA was extracted. Based on the S gene sequences of Hantaan virus strain 76-118 in GenBank database, specific primers were designed based on the above experiments. Total RNA was used as the detection sample to establish qRT-PCR for rapid and quantitative detection of neutralizing antibody activity in Hantaan virus infection experimental method. RESULTS AND CONCLUSION: A modified method of qRT-PCR based microtiter cell neutralization assay was established to quantitatively determine the neutralizing antibody activity in Hantaan virus infection. The method is rapid, sensitive, specific and reproducible.