Liu Baoyuan, Zhao Qin, Sun Yani, Wang Xinjie, Zhao Jinan, Du Taofeng, Wang Chengbao, Xiao Shuqi, Mu Yang, Zhang Gaiping, Luo Jianxun, Hsu Walter H, Zhou En-Min
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, China Ministry of Agriculture, Yangling, Shaanxi 712100, China.
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China.
J Virol Methods. 2014 Aug;204:1-5. doi: 10.1016/j.jviromet.2014.03.023. Epub 2014 Apr 13.
A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (HEV). In the bELISA, the coating antigen was a truncated protein containing C-terminal 268-amino acid region of ORF2 from an avian HEV strain isolated in China (CaHEV) and blocking antibody was a monoclonal antibody (mAb) 1H5 recognizing the epitope within amino acids 384-414 in the C-terminal 268-amino acid region. The concentration of blocking mAb 1H5 was determined as that yielded an OD450nm value of 1.0 for binding to the coating antigen and the antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 20.7% at the mean percent inhibition plus 3 standard deviations was determined by testing 265 negative sera. The bELISA had a sensitivity of 98.3% by testing 116 positive sera from chickens infected experimentally with CaHEV and had no cross-reaction with other anti-avian virus antibodies. The compliance rates of the bELISA with indirect ELISA and Western blot were 83.7% and 93.3%, respectively, by testing 300 field chicken sera. These results suggested that the bELISA developed in this study can be used for detection of antibodies against avian HEV and showed high reproducibility compared with indirect ELISA and Western blot methods.
开发了一种阻断酶联免疫吸附测定法（bELISA）用于检测抗禽戊型肝炎病毒（HEV）的免疫球蛋白G抗体。在该bELISA中，包被抗原是一种截短蛋白，包含来自中国分离的禽HEV毒株（CaHEV）的ORF2的C端268个氨基酸区域，阻断抗体是一种单克隆抗体（mAb）1H5，可识别C端268个氨基酸区域中氨基酸384 - 414内的表位。确定阻断mAb 1H5的浓度，使其与包被抗原结合时产生的OD450nm值为1.0，并使用棋盘滴定法优化抗原浓度和血清稀释度。通过检测265份阴性血清，确定平均抑制百分比加3个标准差时的临界值为20.7%。通过检测116份经CaHEV实验感染鸡的阳性血清，该bELISA的灵敏度为98.3%，且与其他抗禽病毒抗体无交叉反应。通过检测300份田间鸡血清，该bELISA与间接ELISA和Western blot的符合率分别为83.7%和93.3%。这些结果表明，本研究中开发的bELISA可用于检测抗禽HEV抗体，与间接ELISA和Western blot方法相比具有较高的重复性。
