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目的 为了建立裂谷热病毒(Rift Valley fever virus, RVFV)核蛋白双抗体夹心ELISA检测方法。方法 本研究以真核表达的RVFV核蛋白作为抗原，选用4株鼠源抗RVFV NP蛋白单克隆抗体(Monoclonal antibodies,mAbs)及其辣根过氧化酶(Horseradish peroxidase, HRP)标记抗体分别作为捕获抗体和检测抗体进行最佳抗体配对筛选。采用棋盘格滴定法优化反应条件，建立RVFV核蛋白双抗体夹心ELISA检测方法，并对其敏感性、特异性、重复性和模拟临床样本的检测进行评估。结果 结果显示，该方法对灭活RVFV的最低检测限为101.78 TCID50/mL，对真核表达核蛋白的最低检测限为25.6095 ng/mL。特异性试验结果显示，该检测方法仅能检测灭活RVFV样本，与埃博拉病毒(Ebola virus, EBOV)、发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus, SFTSV)、新疆出血热病毒(Xinjiang hemorrhagic fever virus, XHFV)的NP蛋白以及鼠伤寒沙门菌(S. Typhimurium)的O抗原均无交叉反应。重复性试验结果显示，批内和批间变异系数均小于10%，表明该检测方法具有良好的重复性。模拟临床样本检测结果显示，该方法与金标准Real-time RT-PCR检测结果的符合率为100%，具有良好的临床应用潜力。结论 综上所述，本研究成功建立了一种灵敏、特异且稳定的RVFV核蛋白双抗体夹心ELISA检测方法，为裂谷热病毒的早期检测与流行病学监测提供了可靠的技术支持。
Abstract：Objective To establish and evaluate a double-antibody sandwich ELISA for the detection of Rift Valley fever virus(RVFV) nucleoprotein(NP). Methods Eukaryotically expressed RVFV NP was used as the antigen. Four murine monoclonal antibodies(mAbs) against RVFV NP and their corresponding horseradish peroxidase(HRP)-conjugated antibodies were evaluated as capture and detection antibodies to identify the optimal antibody pair. Reaction conditions were optimized by checkerboard titration. The established assay was subsequently assessed for sensitivity, specificity, reproducibility, and performance using simulated clinical samples. Results The detection limit of the assay was 101.78 TCID50/mL for inactivated RVFV and 25.61 ng/mL for recombinant RVFV NP. Specificity testing demonstrated that the assay detected only inactivated RVFV and showed no cross-reactivity with the nucleoproteins of Ebola virus(EBOV), severe fever with thrombocytopenia syndrome virus(SFTSV), Xinjiang hemorrhagic fever virus(XHFV), or the O antigen of Salmonella enterica serovar Typhimurium. Both intra-assay and inter-assay coefficients of variation were below 10%, indicating good reproducibility. In simulated clinical samples, the ELISA showed 100% concordance with Real-time RT-PCR, suggesting strong potential for clinical application. Conclusion A sensitive, specific, and reproducible double-antibody sandwich ELISA targeting RVFV nucleoprotein was successfully developed. This assay provides a reliable tool for early detection and epidemiological surveillance of Rift Valley fever virus.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250420
中图分类号:S852.65
引用信息:
[1]张子莫,栾娇彦,李昊,等.裂谷热病毒核蛋白双抗体夹心ELISA检测方法的建立与评价[J].病毒学报,2026,42(02):592-600.DOI:10.13242/j.cnki.bingduxuebao.250420.
基金信息:
“十四五”国家重点研发计划青年科学家项目（项目号：2021YFF0703600）,题目：实验动物病原快速检测新技术研究; 吉林省科技发展计划重点研发项目（项目号：20250202061NC）,题目：裂谷热病毒快速检测技术研究~~
2025-12-29
2025
2026-03-09
2026
2
2026-03-12
2026-03-12
2026-03-12