Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993, USA.
Viruses. 2024 Mar 2;16(3):393. doi: 10.3390/v16030393.
Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by genotypes 1-4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin-streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2-3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.
戊型肝炎病毒 (HEV) 可引起人类急性肝炎，在免疫抑制个体中可进展为慢性肝炎。几乎所有报告的 HEV 感染均由基因型 1-4 引起。结构 ORF2 蛋白是 HEV 感染个体血液中检测到的主要抗原。用于检测 HEV IgM 抗体的 ELISA 检测法是一线诊断检测法；然而，这些检测法的表现存在差异，结果经常不一致。目前有一种用于研究的定性 HEV 抗原（ORF2）ELISA。在这里，我们报告了一种新的定量夹心 ELISA，用于测量 3 种基质类型中的 HEV ORF2 蛋白。在测试的 12 种独特组合中，选择了最佳的捕获和检测抗体对。使用这些单克隆抗体和生物素-链霉亲和素技术开发了夹心 ELISA 方案。通过从标准曲线内插，进一步优化了方案，以在 3.17 至 50.8 飞摩尔/毫升的线性范围内定量不同基质中的 ORF2 抗原。使用该方法，早在用 HEV 基因组 RNA 转染 Huh7 细胞后 2-3 天，以及感染 HEV 的人肝细胞培养基中，就可检测到细胞培养物中的 ORF2 蛋白。在感染 HEV 后的前 2 周，即可在沙土鼠血清中轻易检测到 ORF2 抗原。在免疫抑制的沙土鼠中，可检测到 ORF2 长达 6 周，感染后 3 至 6 周的水平明显更高。HEV ORF2 抗原水平与细胞培养物和沙土鼠血清中的 HEV RNA 水平呈强正相关。我们的新型夹心 ELISA 可检测到人血浆中用细胞培养物传播的 HEV 感染的至少 7.3 飞摩尔/毫升 ORF2 蛋白，并可检测到 HEV RNA 阳性但抗 HEV 抗体阴性的人血浆样品中的 ORF2 蛋白。此外，该检测法对人阴性血浆和 HBV 或 HCV 阳性人血浆无反应，显示出特异性。总的来说，我们的 ORF2 抗原 ELISA 将有助于定量细胞培养物、沙土鼠血清和人血浆中的 ORF2 抗原。需要进一步的研究来评估其在 HEV 临床诊断中的应用。
