Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling, 712100, Shaanxi, China.
Appl Microbiol Biotechnol. 2021 Nov;105(21-22):8505-8516. doi: 10.1007/s00253-021-11621-3. Epub 2021 Oct 11.
Hepatitis E, a significant global public health issue in China, is caused by sporadic infections with regional hepatitis E virus (HEV) genotypes 1, 3, and 4. To date, most immunoassays currently used to test human sera for the presence of anti-HEV antibodies cannot identify HEV at the genotype level. However, such information would be useful for identifying the source of infecting virus. Therefore, here we describe the development of a competitive enzyme-linked immunosorbent assay (ELISA) for detecting anti-genotype 1 HEV antibodies in human sera. Using recombinant genotype 1 HEV ORF3 protein as immunogen, traditional hybridoma technology was employed to generate seven monoclonal antibodies (mAbs), of which two mAbs specifically reacted with the immunogen. One of these two mAbs, 1D2, was labeled with horseradish peroxidase (HRP) for use in competitive ELISA (cELISA). After cELISA optimization using a checkerboard assay design, the amount of ORF3 as coating antigen (100 ng/well), HRP-1D2 mAb concentration (1 μg/mL), and test serum dilution (1:10) were selected and a result ≥ 19.5 was used as the cutoff for a positive result. Importantly, cross-genotype cELISA results indicated that the cELISA could not detect anti-genotype 3 rabbit and 4 swine HEV antibodies. Moreover, human sera confirmed as negative for anti-HEV antibodies using the commercial ELISA kit were all negative via cELISA. However, because the commercial ELISA kit detects anti-all genotypes HEV antibodies and the cELISA only detects anti-genotype 1 HEV antibodies, the consistence rate of two assays detecting positive sera is low. In summary, here a cELISA for detecting anti-genotype 1 HEV antibodies was developed for use in epidemiological investigations of genotype 1 HEV infections in humans. KEY POINTS: • Seven mAbs were produced using genotype 1 HEV ORF3 protein as immunogen. • One mAb that specifically bound to genotype 1 HEV ORF3 protein was selected and labeled for use in a cELISA to detect anti-genotype 1 HEV antibodies. • The competitive ELISA developed here will aid clinical diagnosis of HEV infections and will be useful for large-scale serological testing of genotype 1 HEV infections in humans.
戊型肝炎，中国重要的全球性公共卫生问题，是由散发感染具有地域特征的戊型肝炎病毒（HEV）基因型 1、3 和 4 引起的。迄今为止，大多数用于检测人血清中抗-HEV 抗体的免疫测定法目前无法在基因型水平上检测 HEV。然而，这些信息对于确定感染病毒的来源将很有用。因此，我们在这里描述了一种用于检测人血清中抗-1 型 HEV 抗体的竞争酶联免疫吸附测定法（ELISA）的开发。使用重组基因型 1 HEV ORF3 蛋白作为免疫原，采用传统的杂交瘤技术产生了 7 株单克隆抗体（mAbs），其中 2 株 mAbs 特异性地与免疫原反应。这 2 株 mAbs 中的一株，1D2，被辣根过氧化物酶（HRP）标记，用于竞争 ELISA（cELISA）。使用棋盘试验设计对 cELISA 进行优化后，选择了作为包被抗原的 ORF3 量（100ng/孔）、HRP-1D2 mAb 浓度（1μg/mL）和测试血清稀释度（1:10），结果≥19.5 被用作阳性结果的截止值。重要的是，交叉基因型 cELISA 结果表明，cELISA 不能检测抗-3 型兔和 4 型猪 HEV 抗体。此外，使用商业 ELISA 试剂盒确认为抗-HEV 抗体阴性的人血清在 cELISA 中均为阴性。然而，由于商业 ELISA 试剂盒检测抗所有基因型 HEV 抗体，而 cELISA 仅检测抗-1 型 HEV 抗体，因此两种检测方法检测阳性血清的一致性率较低。总之，这里开发了一种用于检测 1 型 HEV 抗体的 cELISA，用于人类 1 型 HEV 感染的流行病学调查。关键点： • 使用基因型 1 HEV ORF3 蛋白作为免疫原产生了 7 株 mAbs。 • 选择并标记了一种特异性结合基因型 1 HEV ORF3 蛋白的 mAb，用于 cELISA 检测抗-1 型 HEV 抗体。 • 这里开发的竞争性 ELISA 将有助于戊型肝炎感染的临床诊断，并将有助于对人类 1 型 HEV 感染进行大规模血清学检测。
