Zhang Mengran, Wei Xinyu, Qian Jing, Yu Zhengyu, Liu Xin, Luo Yan, Zhang Haitao, Gu Youfang, Li Yin
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing 210014, China.
College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China.
Vaccines (Basel). 2023 Mar 15;11(3):664. doi: 10.3390/vaccines11030664.
Goose astrovirus (GAstV) was classified into GAstV-1 and GAstV-2, and both caused gosling viral gout. Recently, there has been no effective commercial vaccine to control the infection. It is important to establish serological methods to distinguish between the two genotypes. In this study, we reported the development and application of two indirect enzyme-linked immunosorbent assays (ELISAs) using the GAstV-1 virus and a recombinant GAstV-2 capsid protein as specific antigens to detect antibodies against GAstV-1 and GAstV-2, respectively. The optimal coating antigen concentration of indirect GAstV-1-ELISA and GAstV-2-Cap-ELISA was 1.2 µg/well and 125 ng/well, respectively. In addition, the antigen coating temperature and time, sera dilution and reaction time, and the dilution and reaction time of HRP-conjugated secondary antibody were optimized. The cut-off values were 0.315 and 0.305, and the analytical sensitivity was 1:6400 and 1:3200 for indirect GAstV-1-ELISA and GAstV-2-Cap-ELISA, respectively. The assays were able to differentiate specific sera against GAstVs, TUMV, GPV, and H9N2-AIV. The intra- and inter-plate variabilities of indirect ELISAs were less than 10%. The coincidence rate of positive sera was higher than 90%. The indirect ELISAs were further applied to test 595 goose serum samples. The results showed that the detection rates were 33.3% and 71.4% in GAstV-1-ELISA and GAstV-2-Cap-ELISA, respectively, and the co-detection rate was 31.1%, which indicates that the seroprevalence rate of GAstv-2 was higher than that of GastV-1, and the co-infection existed between GAstV-1 and GAstV-2. In summary, the developed GAstV-1-ELISA and GAstV-2-Cap-ELISA have high specificity, sensitivity, and reproducibility and can be used in the clinical detection of the antibody against GAstV-1 and GAstV-2.
鹅星状病毒（GAstV）分为GAstV - 1和GAstV - 2，二者均可引起雏鹅病毒性痛风。目前，尚无有效的商业疫苗来控制该感染。建立血清学方法区分这两种基因型很重要。在本研究中，我们报道了两种间接酶联免疫吸附测定（ELISA）的建立及应用，分别以GAstV - 1病毒和重组GAstV - 2衣壳蛋白作为特异性抗原，用于检测抗GAstV - 1和抗GAstV - 2的抗体。间接GAstV - 1 - ELISA和GAstV - 2 - Cap - ELISA的最佳包被抗原浓度分别为1.2 μg/孔和125 ng/孔。此外，还优化了抗原包被温度和时间、血清稀释度和反应时间以及辣根过氧化物酶（HRP）标记二抗的稀释度和反应时间。间接GAstV - 1 - ELISA和GAstV - 2 - Cap - ELISA的临界值分别为0.315和0.305，分析灵敏度分别为1:6400和1:3200。这些检测方法能够区分抗GAstV、烟草花叶病毒（TUMV）、鹅细小病毒（GPV）和H9N2禽流感病毒（H9N2 - AIV）的特异性血清。间接ELISA的板内和板间变异系数均小于10%。阳性血清的符合率高于90%。间接ELISA进一步应用于检测595份鹅血清样本。结果显示，GAstV - 1 - ELISA和GAstV - 2 - Cap - ELISA的检出率分别为33.3%和71.4%，共同检出率为31.1%，这表明GAstV - 2的血清流行率高于GAstV - 1，且GAstV - 1和GAstV - 2之间存在共同感染。综上所述，所建立的GAstV - 1 - ELISA和GAstV - 2 - Cap - ELISA具有高特异性、灵敏度和重复性，可用于临床检测抗GAstV - 1和抗GAstV - 2的抗体。
