Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAVs infection in different species. Recently, nanobodies, which show advantages of easy gene-editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can cross-react with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen, as well as dilutions of the fusion and testing sera were 100 ng/well, 1:4,000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. Additionally, we found that the cELISA and hemagglutination inhibition (HI) test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species.

甲型流感病毒 (IAV) 的传播，尤其是在家禽和猪体内的传播，继续威胁着公众健康。一种简单通用的检测方法对于监测不同物种的 IAVs 感染非常重要。最近，纳米抗体显示出易于基因编辑和生产成本低的优势，是一种很有前途的新型诊断工具，用于监测和控制全球 IAV。在本研究中，通过噬菌体展示从免疫的双峰驼中筛选出 5 个针对 H9N2 IAV 核蛋白的纳米抗体，并用辣根过氧化物酶 (HRP) 标签进行修饰。其中，我们确定 H9N2-NP-Nb5-HRP 可以与 IAV 的不同亚型发生交叉反应，并且这种反应也被针对不同 IAV 亚型的抗体阳性血清所阻断。表位作图显示纳米抗体-HRP 融合识别了所有 IAV 亚型中的保守构象表位。随后，我们开发了一种基于纳米抗体的竞争性 ELISA（cELISA），用于检测不同物种的抗 IAV 抗体。包被抗原的优化量以及融合和测试血清的稀释度分别为 100 ng/孔、1:4,000 和 1:10。操作 cELISA 的时间约为 35 分钟。cELISA 显示出高灵敏度、特异性、重现性和稳定性。此外，我们发现 cELISA 和血凝抑制 (HI) 测试显示临床和攻击鸡血清的一致性分别为 100% 和 87.91%。此外，cELISA 和商业 IEDXX ELISA 试剂盒之间的一致率分别为 90.4% 和 85.7%。总的来说，