Institute of Ecology and Genetics of Microorganisms, Ural Branch of Russian Academy of Sciences, 614081 Perm, Russia.
Biology Faculty, Perm State University, 614990 Perm, Russia.
Biosensors (Basel). 2023 Aug 29;13(9):857. doi: 10.3390/bios13090857.
Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5-7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.
即时检测在传染病血清学诊断和疫苗接种后免疫监测中发挥着重要作用，包括在 COVID-19 中的作用。目前，侧向流动检测在该领域占据主导地位，具有良好的分析性能。然而，提高此类检测效果的研究仍然很重要。与侧向流动检测相比，垂直流免疫分析可以缩短检测时间并减少钩状效应的影响。此外，使用碳黑纳米粒子（CNP）作为颜色标记可以比传统胶体金提供更低的检测限（LOD）。因此，我们通过应用与抗人 IgG 单克隆抗体（CNP@MAb）偶联的 CNP 开发了一种用于检测人血清样品中针对 SARS-CoV-2 刺突蛋白 IgG 的垂直流免疫分析。垂直流分析装置由一个顶部有孔的塑料盒组成，其中包含涂有刺突蛋白的硝酸纤维素膜和吸收垫。血清样品、洗涤缓冲液和 CNP@MAb 垂直流过硝酸纤维素膜和吸收垫，从而缩短了检测时间并简化了操作程序。在阳性样本中，CNP@MAb 与抗刺突抗体的相互作用导致黑色斑点的出现，可以通过肉眼观察到。所开发的方法可以快速（5-7 分钟）进行 IgG 与刺突蛋白的可视化检测，LOD 为 7.81 BAU/mL。结果表明，未经训练的操作人员可以进行检测并对结果进行目视评估。因此，所提出的检测方法可用于进一步开发 COVID-19 血清学诊断或疫苗接种后免疫监测的检测系统。
