Abstract

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.

中文翻译：

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单克隆抗体的生产和鉴定，以及用于检测H3亚型禽流感病毒抗原的夹心ELISA的开发

摘要

H3亚型禽流感病毒（AIV）广泛存在于禽类中，并经常在监视项目中分离；因此，我们开发了一种基于单克隆抗体（mAb）的夹心ELISA用于H3 AIV检测的更有效的诊断方法。首先，通过开发纯化的H3亚型AIV菌株的mAb小鼠免疫接种和细胞融合以产生杂交瘤细胞的方法，我们生产了抗AIV H3菌株的mAb必需试剂。用血凝抑制（HI）试验筛选这些细胞，并亚克隆最佳细胞。我们选择了稳定分泌特异的H3亚型AIV mAb（称为9F12）的杂交瘤细胞系，该抗体属于IgG1亚类并具有K型轻链。通过免疫荧光测定和蛋白质印迹分析，显示9F12与H3-亚型AIV抗原特异性结合。最后，成功开发了基于9F12的夹心ELISA，并用于特异性测试该抗原。优化了夹心ELISA条件，并验证了特异性和敏感性。临床样品检测结果与病毒分离结果一致。因此，基于9F12的夹心ELISA是一种针对H3亚型AIV的特异性，灵敏，鲁棒，快速且通用的诊断工具，为有效预防和控制流感病毒提供了有希望的策略。并验证了特异性和敏感性。临床样品检测结果与病毒分离结果一致。因此，基于9F12的夹心ELISA是一种针对H3亚型AIV的特异性，灵敏，鲁棒，快速且通用的诊断工具，为有效预防和控制流感病毒提供了有希望的策略。并验证了特异性和敏感性。临床样品检测结果与病毒分离结果一致。因此，基于9F12的夹心ELISA是一种针对H3亚型AIV的特异性，灵敏，鲁棒，快速且通用的诊断工具，为有效预防和控制流感病毒提供了有希望的策略。