PDF(Pubmed)
Abstract：
BACKGROUND: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies.
METHODS: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron.
RESULTS: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies.
CONCLUSIONS: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.
METHODS: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron.
RESULTS: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies.
CONCLUSIONS: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.
摘要：
背景：由严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）引起的COVID-19，现已成为地方病，是目前经常影响人类的重要呼吸道病毒感染之一。对SARS-CoV-2及其变体的免疫评估对于指导主动和被动免疫以及SARS-CoV-2特异性治疗策略很重要。
方法：我们设计了一种新的基于流式细胞术的诊断平台，用于评估针对细胞结合病毒抗原的免疫力。该平台基于HEK-293T细胞系的集合，正如我们研究中的例子，稳定表达八种主要SARS-CoV-2变体的SARS-CoV-2S蛋白的受体结合域（RBD），从武汉-胡-1到奥米克。
结果：表达RBD的细胞系在HEK-293T细胞表面稳定地显示出相当水平的RBD，如针对N末端引入的3x-FLAG序列的抗FLAG标签抗体所示,而RBD的功能性通过ACE2结合得到证实。我们通过直接结合暴露于SARS-CoV-2和/或接种疫苗的个体的IgG和IgA抗体来举例说明基于细胞的测试的有用性和特异性,其中该测定在非常低和非常高的血清抗体浓度下显示宽的线性性能范围。在另一个应用中，即，抗体吸附研究，该测试被证明是测量单个变体特异性抗体比率的强大工具。
结论：我们已经建立了一个工具箱，用于测量针对细胞结合病毒抗原的SARS-CoV-2特异性免疫，这可能被认为是SARS-CoV-2特异性诊断测试的重要补充，允许灵活和快速适应新的变种的关注。
方法：我们设计了一种新的基于流式细胞术的诊断平台，用于评估针对细胞结合病毒抗原的免疫力。该平台基于HEK-293T细胞系的集合，正如我们研究中的例子，稳定表达八种主要SARS-CoV-2变体的SARS-CoV-2S蛋白的受体结合域（RBD），从武汉-胡-1到奥米克。
结果：表达RBD的细胞系在HEK-293T细胞表面稳定地显示出相当水平的RBD，如针对N末端引入的3x-FLAG序列的抗FLAG标签抗体所示,而RBD的功能性通过ACE2结合得到证实。我们通过直接结合暴露于SARS-CoV-2和/或接种疫苗的个体的IgG和IgA抗体来举例说明基于细胞的测试的有用性和特异性,其中该测定在非常低和非常高的血清抗体浓度下显示宽的线性性能范围。在另一个应用中，即，抗体吸附研究，该测试被证明是测量单个变体特异性抗体比率的强大工具。
结论：我们已经建立了一个工具箱，用于测量针对细胞结合病毒抗原的SARS-CoV-2特异性免疫，这可能被认为是SARS-CoV-2特异性诊断测试的重要补充，允许灵活和快速适应新的变种的关注。
