Li Jia, Yang Yuhang, Jia Kuangrou, Zhang Zhigao, Zhai Xiangmin, Cao Yue, Huang Xinbiao, Cao Shouchun, Wu Yingsong, Lin Guanfeng
National Institutes for Food and Drug Control, Beijing, China.
State Key Laboratory of Drug Regulatory Science, Beijing, China.
PLoS Negl Trop Dis. 2025 May 30;19(5):e0013077. doi: 10.1371/journal.pntd.0013077. eCollection 2025 May.
Currently, the integrity of rabies virus particle in quality control can only be assessed through electron microscopy. However, its time-consuming nature, operational complexity, limited accuracy and lack of quantification capability no longer meet the needs of modern vaccine production. Based on this, we developed a time-resolved fluoroimmunoassay (TRFIA) to enable real-time quantitative analysis of rabies virus particle integrity in human rabies vaccine, by detecting exposed nucleoprotein. Monoclonal antibodies against rabies glycoprotein and nucleoprotein were prepared using the classical hybridoma technology with the aim of constructing a novel detection approach for assessing particle integrity. A monoclonal antibody against rabies glycoprotein was immobilized on microplate wells to capture rabies virus particles, while a labeled antibody against the nucleoprotein served as the signal tracer. Multiple types of vaccine samples were analyzed to evaluate the effectiveness and accuracy of the developed TRFIA, combined with various virus particle destruction methods to validate its capability to assess particle integrity. The validation results were consistent with those obtained from electron microscopy. Therefore, this novel TRFIA offers a promising solution to address existing gaps in current analytical methods, enabling straightforward real-time, and quantitative evaluation of rabies virus particle integrity in the laboratory.
目前，狂犬病病毒颗粒质量控制中的完整性只能通过电子显微镜进行评估。然而，其耗时的特性、操作复杂性、有限的准确性以及缺乏定量能力已不再满足现代疫苗生产的需求。基于此，我们开发了一种时间分辨荧光免疫分析法（TRFIA），通过检测暴露的核蛋白，对人用狂犬病疫苗中的狂犬病病毒颗粒完整性进行实时定量分析。利用经典的杂交瘤技术制备了抗狂犬病糖蛋白和核蛋白的单克隆抗体，旨在构建一种评估颗粒完整性的新型检测方法。将抗狂犬病糖蛋白的单克隆抗体固定在微孔板孔中以捕获狂犬病病毒颗粒，而抗核蛋白的标记抗体用作信号示踪剂。结合多种病毒颗粒破坏方法，对多种类型的疫苗样品进行分析，以评估所开发的TRFIA的有效性和准确性，验证其评估颗粒完整性的能力。验证结果与电子显微镜获得的结果一致。因此，这种新型的TRFIA为解决当前分析方法中存在的差距提供了一个有前景的解决方案，能够在实验室中直接进行实时、定量评估狂犬病病毒颗粒的完整性。
