Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Kashan University of Medical Sciences, Kashan, Iran.
Viral Immunol. 2023 Jul-Aug;36(6):378-388. doi: 10.1089/vim.2022.0196. Epub 2023 Jun 9.
Hepatitis B virus (HBV) infection is a major health problem worldwide and causes almost one million deaths annually. The HBV core gene codes for two related antigens, known as core antigen (HBcAg) and e-antigen (HBeAg), sharing 149 residues but having different amino- and carboxy-terminals. HBeAg is a soluble variant of HBcAg and a clinical marker for determining the disease severity and patients' screening. Currently available HBeAg assays have a shortcoming of showing cross-reactivity with HBcAg. In this study, for the first time, we evaluated whether HBcAg-adsorbed anti-HBe polyclonal antibodies could specifically recognize HBeAg or still show cross-reactivity with HBcAg. Recombinant HBeAg was cloned in pCold1 vector and successfully expressed in and after purification by Ni-NTA resin was used to generate polyclonal anti-HBe antibodies in rabbit. Purified HBeAg was further characterized by assessing its reactivity with anti-HBe in the sera of chronically infected patients and HBeAg-immunized rabbit. Sera from patients with chronic HBV infection, containing anti-HBe, specifically reacted with recombinant HBeAg, implying antigenic similarity between the prokaryotic and native HBeAg in the serum of HBV-infected patients. In addition, the designed enzyme-linked immunosorbent assay (ELISA) with rabbit anti-HBe polyclonal antibodies could detect recombinant HBeAg with high sensitivity, while high cross-reactivity with HBcAg was observed. It is noteworthy that HBcAg-adsorbed anti-HBe polyclonal antibodies still showed high cross-reactivity with HBcAg, implying that due to the presence of highly similar epitopes in both antigens, HBcAg-adsorbed polyclonal antibodies cannot differentiate between the two antigens.
乙型肝炎病毒 (HBV) 感染是全球范围内的一个主要健康问题，每年导致近百万人死亡。HBV 核心基因编码两种相关抗原，分别称为核心抗原 (HBcAg) 和 e 抗原 (HBeAg)，它们共享 149 个残基，但具有不同的氨基和羧基末端。HBeAg 是 HBcAg 的可溶性变体，是确定疾病严重程度和患者筛查的临床标志物。目前可用的 HBeAg 检测方法存在与 HBcAg 交叉反应的缺点。在这项研究中，我们首次评估了 HBcAg 吸附的抗-HBe 多克隆抗体是否可以特异性识别 HBeAg，还是仍然与 HBcAg 发生交叉反应。重组 HBeAg 被克隆到 pCold1 载体中，并在 中成功表达，然后通过 Ni-NTA 树脂纯化，用于在兔中产生抗-HBe 多克隆抗体。通过评估其与慢性感染患者血清中的抗-HBe 以及 HBeAg 免疫兔血清中的反应性，进一步对纯化的 HBeAg 进行了表征。来自慢性 HBV 感染患者的血清，含有抗-HBe，特异性地与重组 HBeAg 反应，这表明在 HBV 感染患者血清中，原核和天然 HBeAg 之间存在抗原相似性。此外，使用兔抗-HBe 多克隆抗体设计的酶联免疫吸附试验 (ELISA) 可以高灵敏度检测重组 HBeAg，同时与 HBcAg 表现出高交叉反应性。值得注意的是，HBcAg 吸附的抗-HBe 多克隆抗体仍与 HBcAg 表现出高交叉反应性，这表明由于两种抗原中存在高度相似的表位，HBcAg 吸附的多克隆抗体无法区分这两种抗原。
