Vertical flow immunoassay based on carbon black nanoparticles for the detection of IgG against SARS-CoV-2 Spike-protein in human serum: proof-of-concept

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including 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 (CNP) 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 CNP 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 the 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 minutes) 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.