Chen Yiyang, Zhao Qin, Liu Baoyuan, Wang Lizhen, Sun Yani, Li Huixia, Wang Xinjie, Syed Shahid Faraz, Zhang Gaiping, Zhou En-Min
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
Scientific Observing and Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture, Yangling, Shaanxi, China.
PLoS One. 2016 Mar 29;11(3):e0152639. doi: 10.1371/journal.pone.0152639. eCollection 2016.
Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.
戊型肝炎病毒（HEV）可感染人类和动物，总体人类死亡率一般低于1%，但在孕妇中高达20%。HEV毒株分为4种主要基因型。人畜共患的基因型3和4与许多工业化国家的散发性人类和动物HEV病例相关。迄今为止，已有多项证据表明猪是主要的HEV宿主，这也证明了监测猪群中HEV感染率以预防人类疾病的重要性。由于缺乏用于病毒繁殖的强大体外细胞培养系统，目前尚未开发出用于检测家猪HEV感染的“金标准”检测方法。1E4是一种单克隆抗体（mAb），对HEV基因型4 ORF2衣壳蛋白（sORF2-C）的C端268个氨基酸具有特异性，将其与辣根过氧化物酶（HRP）偶联，用于阻断ELISA（bELISA）。使用棋盘滴定试验确定了最佳的sORF2-C包被抗原浓度（8μg/ml）、HRP-1E4稀释度（1:1000）和待测猪血清稀释度（1:20）。在对230份阴性猪血清进行平均抑制百分比（PI）检测后，选择16.9%的临界值来区分阳性和阴性血清。与间接ELISA（iELISA）、western印迹法以及用于检测人血清中抗HEV抗体的商业ELISA试剂盒相比，bELISA无统计学差异，与其他检测方法的统计学一致性分别为93.23%、92%和95%。开发了一种用于检测猪血清样本中抗HEV抗体的阻断ELISA（bELISA），其灵敏度和特异性与间接ELISA相当。bELISA结果与iELISA、western印迹法以及用于检测人抗HEV抗体的商业ELISA试剂盒高度一致。因此，bELISA应成为家猪抗HEV抗体大规模血清学调查的理想方法。
