Sun Xuyang, Lv Qingyu, Huang Wenhua, Zhang Xinran, Duan Huiqi, Ren Yuhao, Zhang Xiaojing, Jiang Yongqiang, Zhao Ruili, Chen Shaolong
Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China.
State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China.
Viruses. 2025 May 23;17(6):748. doi: 10.3390/v17060748.
Nipah virus (NiV) is an emerging zoonotic pathogen whose surface glycoprotein (G)-mediated host cell invasion mechanism leads to fatal encephalitis in infected patients (case fatality rate 40-75%). Given the limitations of existing diagnostic technologies, such as low sensitivity and prolonged processing times, we prepared an anti-NiV-G monoclonal antibody to establish a novel Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) detection system. Firstly, five high-affinity anti-NiV-G monoclonal antibodies were screened from the spleens of immunized mice by flow cytometry-single-cell cloning technology. The reaction system was further optimized, and the optimal dilution ratio of antibody-conjugated receptor microspheres, biotinylated antibodies, and donor microspheres was screened, and the AlphaLISA detection platform was successfully constructed. The detection sensitivity of NiV-G protein was 0.024 ng/mL (41.7 times higher than that of conventional ELISA), the coefficient of variation was <9.5%, and the repetition was good. It showed good specificity in the detection of 5 zoonotic viruses, including Japanese encephalitis virus and Zika virus. At the same time, this method is less disturbed by human serum, and the detection time is less than 30 min, showing a good clinical application prospect.
尼帕病毒（NiV）是一种新出现的人畜共患病原体，其表面糖蛋白（G）介导的宿主细胞入侵机制可导致感染患者发生致命性脑炎（病死率40%-75%）。鉴于现有诊断技术存在局限性，如灵敏度低和处理时间长，我们制备了一种抗NiV-G单克隆抗体，以建立一种新型的放大发光邻近均相分析（AlphaLISA）检测系统。首先，通过流式细胞术-单细胞克隆技术从免疫小鼠脾脏中筛选出5种高亲和力抗NiV-G单克隆抗体。进一步优化反应体系，筛选抗体偶联受体微球、生物素化抗体和供体微球的最佳稀释比例，成功构建了AlphaLISA检测平台。NiV-G蛋白的检测灵敏度为0.024 ng/mL（比传统ELISA高41.7倍），变异系数<9.5%，重复性良好。在检测包括日本脑炎病毒和寨卡病毒在内的5种人畜共患病病毒时显示出良好的特异性。同时，该方法受人体血清干扰较小，检测时间少于30分钟，具有良好的临床应用前景。
