Lin Guanfeng, Huang Hong, Liu Tiancai, He Chunhui, Liu Jianqing, Chen Shaolang, Hou Jingyuan, Ren Zhiqi, Dong Wenqi, Wu Yingsong
Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
Guangzhou Promise Biologics Co., Ltd, No. 1 Wanbao North Street, Panyu District, Guangzhou, China.
J Virol Methods. 2014 Sep;206:89-94. doi: 10.1016/j.jviromet.2014.06.002. Epub 2014 Jun 10.
Sensitive, precise and rapid detection tests are needed in the quality control of rabies vaccine for rabies virus nucleoprotein. Previous studies for quantitation of rabies virus nucleoprotein focused on enzyme-linked immunosorbent assay (ELISA). A novel immunoassay for rapid determination of rabies virus nucleoprotein in rabies vaccine was first established by time-resolved fluoroimmunoassay (TRFIA). Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating in the wells and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was retained after washing, and then adopted treatment with enhancement solution; fluorescence was then measured according to the number of europiumions dissociated. Levels of the rabies virus nucleoprotein were measured in a linear range (5-2500 mEU/mL) with a lower limit of quantitation (0.95 mEU/mL) under optimal conditions. The repeatability, recovery, and linearity of the immunoassay were demonstrated to be acceptable. The correlation coefficient of nucleoprotein values obtained by novel TRFIA method and ELISA method was 0.981. These results showed good correlation and confirmed that this sensitive, precise and rapid TRFIA was feasible and could be more suitable for the quality control in the process of rabies vaccine production than ELISA.
狂犬病疫苗质量控制中需要灵敏、精确且快速的检测试验来检测狂犬病病毒核蛋白。以往对狂犬病病毒核蛋白定量的研究主要集中在酶联免疫吸附测定（ELISA）。首次通过时间分辨荧光免疫分析（TRFIA）建立了一种用于快速测定狂犬病疫苗中狂犬病病毒核蛋白的新型免疫分析方法。基于夹心型免疫分析形式，样品中的分析物被孔中包被的一种单克隆抗体捕获，并被另一种标记有铕螯合物的单克隆抗体“夹在中间”。洗涤后保留免疫复合物，然后用增强溶液处理；然后根据解离的铕离子数量测量荧光。在最佳条件下，狂犬病病毒核蛋白水平在5 - 2500 mEU/mL的线性范围内测定，定量下限为0.95 mEU/mL。该免疫分析的重复性、回收率和线性被证明是可接受的。新型TRFIA方法和ELISA方法获得的核蛋白值的相关系数为0.981。这些结果显示出良好的相关性，并证实这种灵敏、精确且快速的TRFIA是可行的，并且比ELISA更适合狂犬病疫苗生产过程中的质量控制。
