Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, 106, Taiwan.
Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, 106, Taiwan.
Appl Microbiol Biotechnol. 2022 Dec;106(24):8183-8194. doi: 10.1007/s00253-022-12288-0. Epub 2022 Nov 21.
The nucleic acid test is still the standard assessment for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by human infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to supporting the confirmation of disease cases, serological assays are used for the analysis of antibody status and epidemiological surveys. In this study, a single Western blot strip (WBS) coated with multiple Escherichia coli (E. coli)-expressed SARS-CoV-2 antigens was developed for comprehensive studies of antibody profiles in COVID-19 patient sera. The levels of specific antibodies directed to SARS-CoV-2 spike (S), S2, and nucleocapsid (N) proteins were gradually increased with the same tendency as the disease progressed after hospitalization. The signal readouts of S, S2, and N revealed by the multi-antigen-coated WBS (mWBS)-based serological assay (mWBS assay) also demonstrated a positive correlation with the SARS-CoV-2 neutralizing potency of the sera measured by the plaque reduction neutralization test (PRNT) assays. Surprisingly, the detection signals against the unstructured receptor-binding domain (RBD) purified from E. coli inclusion bodies were not observed, although the COVID-19 patient sera exhibited strong neutralizing potency in the PRNT assays, suggesting that the RBD-specific antibodies in patient sera mostly recognize the conformational epitopes. Furthermore, the mWBS assay identified a unique and major antigenic epitope at the residues 1148, 1149, 1152, 1155, and 1156 located within the 1127-1167 fragment of the S2 subunit, which was specifically recognized by the COVID-19 patient serum. The mWBS assay can be finished within 14-16 min by using the automatic platform of Western blotting by thin-film direct coating with suction (TDCS WB). Collectively, the mWBS assay can be applied for the analysis of antibody responses, prediction of the protective antibody status, and identification of the specific epitope. KEY POINTS: • A Western blot strip (WBS) coated with multiple SARS-CoV-2 antigens was developed for the serological assay. • The multi-antigen-coated WBS (mWBS) can be utilized for the simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens. • The mWBS-based serological assay (mWBS assay) identified a unique epitope recognized by the COVID-19 patient serum.
核酸检测仍然是诊断 2019 年冠状病毒病（COVID-19）的标准评估方法，COVID-19 是由人类感染严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）引起的。除了支持疾病病例的确认外，血清学检测还用于分析抗体状态和流行病学调查。在这项研究中，开发了一种带有多个大肠埃希菌（E. coli）表达的 SARS-CoV-2 抗原的单条 Western blot 条（WBS），用于全面研究 COVID-19 患者血清中的抗体谱。住院后，针对 SARS-CoV-2 刺突（S）、S2 和核衣壳（N）蛋白的特异性抗体水平逐渐升高，与疾病进展趋势相同。基于多抗原包被的 WBS（mWBS）的血清学检测（mWBS 检测）所揭示的 S、S2 和 N 的信号读出结果也与通过蚀斑减少中和试验（PRNT）检测测量的血清中 SARS-CoV-2 中和效力呈正相关。令人惊讶的是，尽管 COVID-19 患者血清在 PRNT 检测中表现出很强的中和效力，但从大肠杆菌包涵体中纯化的未折叠受体结合域（RBD）的检测信号未被观察到，这表明患者血清中的 RBD 特异性抗体主要识别构象表位。此外，mWBS 检测鉴定了 S2 亚基 1127-1167 片段内的残基 1148、1149、1152、1155 和 1156 处的独特且主要的抗原表位，该表位由 COVID-19 患者血清特异性识别。mWBS 检测可通过 Western 印迹薄膜直接涂层抽吸自动平台（TDCS WB）在 14-16 分钟内完成。总的来说，mWBS 检测可用于分析抗体反应、预测保护性抗体状态和鉴定特定表位。 关键点： • 开发了一种带有多个 SARS-CoV-2 抗原的 Western blot 条（WBS），用于血清学检测。 • 多抗原包被的 WBS（mWBS）可用于同时检测针对多种 SARS-CoV-2 抗原的抗体反应。 • mWBS 基于血清学检测（mWBS 检测）鉴定了由 COVID-19 患者血清识别的独特表位。
