Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus
Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus
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DOI：
10.1007/s00253-021-11621-3
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发表时间：
2021-10-11
影响因子：
5
通讯作者：
Zhou，En-Min
中科院分区：
文献类型：
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作者：
Zhang，Beibei;Fan，Jie;Zhou，En-Min
戊型肝炎是由区域性戊型肝炎病毒（HEV）基因型1、3和4散发感染引起的，是中国一个重要的全球公共卫生问题。迄今为止，目前用于检测人血清中是否存在抗HEV抗体的大多数免疫测定法不能在基因型水平上识别HEV。但是，这些信息对于确定感染病毒的来源是有用的。因此，我们在这里描述了一种竞争性酶联免疫吸附试验（ELISA）的发展，用于检测人血清中的抗基因型1型HEV抗体。以重组基因型1型HEV ORF3蛋白为免疫原，采用传统杂交瘤技术制备7种单克隆抗体（mab），其中2种单克隆抗体与免疫原发生特异性反应。其中一种单抗1D2用辣根过氧化物酶（HRP）标记，用于竞争性ELISA （cELISA）。采用棋盘格法优化后，选择ORF3SAR−55as包被抗原量（100 ng/孔）、HRP-1D2 mAb浓度（1 μg/mL）、血清稀释度（1:10），以≥19.5为阳性的截止值。重要的是，跨基因型的cELISA结果表明，cELISA不能检测到抗基因3型兔和4型猪HEV抗体。此外，使用商用ELISA试剂盒确认的抗hev抗体阴性的人血清通过cELISA均为阴性。然而，由于商用ELISA试剂盒检测抗所有基因型HEV抗体，而cELISA仅检测抗1基因型HEV抗体，因此两种检测阳性血清的一致性较低。总之，本研究开发了一种检测抗基因1型HEV抗体的cELISA，用于人类基因1型HEV感染的流行病学调查。•以基因型1 HEV ORF3蛋白为免疫原，制备7个单抗。•选择一个特异性结合基因1型HEV ORF3蛋白的单抗，并标记用于cELISA检测抗基因1型HEV抗体。•本研究开发的竞争性ELISA将有助于HEV感染的临床诊断，并可用于人类基因型1型HEV感染的大规模血清学检测。
AbstractHepatitis 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 ORF3SAR−55as 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.