Luo Sisi, Deng Xianwen, Xie Zhixun, Huang Jiaoling, Zhang Minxiu, Li Meng, Xie Liji, Li Dan, Fan Qing, Wang Sheng, Zeng Tingting, Zhang Yanfang, Xie Zhiqin
Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, 51 North Road You Ai, Nanning, 530001, Guangxi, China.
AMB Express. 2020 Mar 14;10(1):49. doi: 10.1186/s13568-020-00988-7.
The H3 subtype of avian influenza virus (AIV) is widespread in avian species and is frequently isolated in surveillance projects; thus, we have developed a more effective diagnostic approach of a monoclonal antibody (mAb)-based sandwich ELISA for the H3 AIV detection. First, we have produced the essential reagent of mAb against AIV H3 strains with the development of an mAb-Mouse immunization with a purified H3-subtype AIV strain and cell fusion to generate hybridoma cells. These cells were screened with hemagglutination inhibition (HI) tests, and optimal cells were subcloned. We chose a hybridoma cell line that steadily secreted a specific H3-subtype AIV mAb, designated 9F12, that belongs to the IgG1 subclass and has a K-type light chain. 9F12 was shown to bind specifically to the H3-subtype AIV antigen by both immunofluorescence assay and Western blot analysis. Finally, a 9F12-based sandwich ELISA was successfully developed and used to specifically test for this antigen. The sandwich ELISA conditions were optimized, and the specificity and sensitivity were validated. The results for clinical sample detection were consistent with viral isolation. Consequently, the 9F12-based sandwich ELISA is a specific, sensitive, robust, rapid and versatile diagnostic tool for H3-subtype AIV and provides a promising strategy for effective influenza virus prevention and control.
禽流感病毒（AIV）的H3亚型在禽类中广泛传播，并且在监测项目中经常被分离出来；因此，我们开发了一种基于单克隆抗体（mAb）的夹心ELISA的更有效的诊断方法来检测H3亚型AIV。首先，我们通过用纯化的H3亚型AIV毒株对小鼠进行免疫并进行细胞融合以产生杂交瘤细胞，制备了针对AIV H3毒株的mAb的关键试剂。这些细胞通过血凝抑制（HI）试验进行筛选，并且对最佳细胞进行亚克隆。我们选择了一个稳定分泌特异性H3亚型AIV mAb的杂交瘤细胞系，命名为9F12，它属于IgG1亚类并且具有K型轻链。通过免疫荧光试验和蛋白质印迹分析表明，9F12能特异性结合H3亚型AIV抗原。最后，成功开发了基于9F12的夹心ELISA并用于特异性检测该抗原。对夹心ELISA的条件进行了优化，并验证了其特异性和敏感性。临床样本检测结果与病毒分离结果一致。因此，基于9F12的夹心ELISA是一种用于H3亚型AIV的特异性、灵敏、稳健、快速且通用的诊断工具，并为有效的流感病毒预防和控制提供了一种有前景的策略。
