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∶为建立发热伴血小板减少综合征病毒（Severe fever with thrombocytopenia syndrome virus, SFTSV）抗原检测方法，本研究基于SFTSV核衣壳蛋白（Nucleocapsid protein, NP）抗原表位分布特征分析，通过大肠杆菌原核表达系统对NP蛋白进行分段重组表达，制备了完整的NP(1～256 aa)、NP氨基端（1～151 aa）和NP蛋白羧基端（149～256 aa）三种蛋白。经Western blot验证重组蛋白免疫原性，免疫新西兰兔制备多克隆抗血清，采用间接ELISA和间接免疫荧光（Immunofluorescence assay, IFA）进行评估和结合活性定量分析，对捕获抗体与酶标抗体最佳组合等反应条件进行优化，比较优化样本处置方式，建立双抗体夹心ELISA检测病毒抗原的方法。以定量的热灭活病毒和重组NP蛋白为参考品绘制标准曲线，评价方法的灵敏性，确定检测限，并通过与qRT-PCR检测确诊SFTS患者血清30份、核酸检测阴性健康体检者血清30份、灭活汉坦病毒和登革病毒各5份、肾综合征出血热患者血清10份，评估方法的其敏感性、特异性和重复性。结果显示，本研究建立的夹心ELISA检测方法对NP蛋白和SFTSV病毒液的最低检出限分别为0.13 pg/μL,17 PFU/100μL，与登革病毒、汉坦病毒不发生交叉反应，批间、批内变异系数均小于5%，样本处理温度和非离子型去污剂的添加是影响抗原检测灵敏度的一个重要因素。综上，本研究建立的双抗体夹心ELISA抗原检测方法具有良好的特异性、灵敏度和重复性，为SFTSV感染的早期临床诊断和现场流行病学调查提供了一种可靠、高效的检测工具。
Abstract：To develop an antigen detection assay for severe fever with thrombocytopenia syndrome virus(SFTSV), we analyzed the epitope distribution characteristics of the viral nucleocapsid protein(NP). Using an Escherichia coli prokaryotic expression system, three recombinant NP proteins were generated by segmented expression∶ full-length NP(1–256 aa), the N-terminal fragment(1–151 aa), and the C-terminal fragment(149 – 256 aa). The immunogenicity of these recombinant proteins was confirmed by Western blot analysis. Polyclonal antisera were then prepared by immunizing New Zealand white rabbits. Indirect ELISA and indirect immunofluorescence assay(IFA) were employed to evaluate and quantify antibody-binding activity. The optimal pairing of capture and enzyme-labeled antibodies was determined, and reaction conditions and sample processing methods were optimized to establish a double-antibody sandwich ELISA for SFTSV antigen detection. Standard curves were generated using quantified heat-inactivated virus and recombinant NP protein as reference materials to assess the analytical sensitivity and establish the limit of detection(LOD). The sensitivity, specificity, and reproducibility of the assay were evaluated using 30 serum samples from SFTSconfirmed patients, 30 sera from nucleic acid – negative healthy controls, 5 samples each of inactivated Hantavirus and Dengue virus, and 10 sera from patients with hemorrhagic fever with renal syndrome. The established sandwich ELISA demonstrated detection limits of 0.13 pg/μL for NP protein and 17 PFU/100 μL for SFTSV, with no cross-reactivity observed against Dengue virus or Hantavirus. Both inter-assay and intraassay coefficients of variation were below 5%. The results indicated that sample processing temperature and the addition of non-ionic detergents were key factors affecting antigen detection sensitivity. In conclusion, the double-antibody sandwich ELISA established in this study shows excellent specificity, sensitivity, and reproducibility, providing a reliable and efficient tool for early clinical diagnosis of SFTSV infection and for field-based epidemiological surveillance.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250243
中图分类号:R373.3
引用信息:
[1]郑岚菱,黄晓霞,杜珊珊,等.双抗体夹心ELISA法检测发热伴血小板减少综合征病毒核蛋白抗原的方法研究[J].病毒学报,2025,41(06):1835-1846.DOI:10.13242/j.cnki.bingduxuebao.250243.
基金信息:
北京市科技计划项目（项目号∶Z221100007422076）,题目∶北京地区呼吸道传染病病原普分析及标准化可组网检测平台应用可行性研究~~
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