Ji Pinpin, Zhang Hao, Yangzong Xiri, Li Ziling, Liu Zhixiang, Dan Miao, Li Xiaoxuan, Sun Xuwen, Zhao Qin, Sun Yani
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China; Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Shaanxi, 712100, China.
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China; Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Shaanxi, 712100, China.
Poult Sci. 2025 Apr;104(4):104970. doi: 10.1016/j.psj.2025.104970. Epub 2025 Mar 4.
Hepatitis-hydropericardium syndrome, caused by fowl adenovirus serotype 4 (FAdV-4), has resulted in significant economic damage to the poultry industry. To monitor viral exposure and vaccine efficacy, some traditional antibody-based immunoassays have been developed for detecting anti-FAdV-4 antibodies. However, these assays have some drawbacks including multi-step operations and higher production cost. Recently, nanobodies are regarded as a promising tool for developing immunoassays. In the study, 23 nanobodies against FAdV-4 were screened and expressed with horseradish peroxidase (HRP) in the HEK293T cells. Then, the FAdV-4-Nb28-HRP fusion protein was selected for developing competitive enzyme-linked immunoassays (cELISA) to detect anti-FAdV-4 antibodies in the chicken sera. The optimal concentrations and dilutions for the coating antigen, fusion protein and testing sera were determined to be 400 ng/well, 1:80 and 1:20, respectively. After the coated plates were vacuumized and stored, the operation of cELISA to detect clinical chicken sera was only one-step and the full time was 75 min. The cELISA also exhibited high sensitivity, specificity, reproducibility and good agreement with the commercial ELISA kit. When the sequential sera from the challenged chickens were tested, the cELISA showed superior sensitivity compared with the commercial ELISA kit. Moreover, epitope mapping revealed that the nanobody specifically recognized the sites GLN235 ASN236 SER238 of the fiber-1 protein, highly conserved among different FAdV-4 isolates and different from the FAdV-1 and -8. The results indicated that cELISA can specifically detect anti-FAdV-4 antibodies. Collectively, the developed one-step nanobody-based cELISA is an ideal method for epidemiological investigation and vaccine immune evaluation of FAdV-4.
由禽腺病毒4型（FAdV-4）引起的肝炎-心包积水综合征给家禽业造成了重大经济损失。为监测病毒暴露情况和疫苗效力，已开发出一些基于传统抗体的免疫测定法来检测抗FAdV-4抗体。然而，这些测定法存在一些缺点，包括操作步骤多和生产成本较高。最近，纳米抗体被视为开发免疫测定法的一种有前景的工具。在该研究中，筛选出了23种抗FAdV-4的纳米抗体，并在HEK293T细胞中与辣根过氧化物酶（HRP）一起表达。然后，选择FAdV-4-Nb28-HRP融合蛋白来开发竞争酶联免疫吸附测定法（cELISA），以检测鸡血清中的抗FAdV-4抗体。包被抗原、融合蛋白和检测血清的最佳浓度和稀释度分别确定为400 ng/孔、1:80和1:20。包被后的酶标板经抽真空和储存后，用于检测临床鸡血清的cELISA操作只需一步，总时长为75分钟。该cELISA还表现出高灵敏度、特异性、重复性，并且与商业ELISA试剂盒具有良好的一致性。当检测受攻毒鸡的连续血清时，cELISA与商业ELISA试剂盒相比表现出更高的灵敏度。此外，表位作图显示该纳米抗体特异性识别纤维-1蛋白的GLN235、ASN236、SER238位点，这些位点在不同的FAdV-4分离株中高度保守，且与FAdV-1和-8不同。结果表明，cELISA能够特异性检测抗FAdV-4抗体。总体而言，所开发的基于纳米抗体的一步法cELISA是FAdV-4流行病学调查和疫苗免疫评估的理想方法。
