Analytical Chemistry 2023
Detection of SARS-CoV-2 N protein. (A) UCNP label: Alkyne-PEG-neridronate strongly binds via two phosphonate groups to surface lanthanide ions of UCNPs, and a click reaction binds the conjugate to azide-modified streptavidin. (B) Scheme of sandwich ULISA: A microtiter plate is coated with two monoclonal antibodies that capture the N protein. Then, two biotinylated detection antibodies bind to the N protein. The sandwich immune complex is finally detected by using the UCNP label.
Significance
The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.
Brandmeier, J.C., Jurga, N., Grzyb, T., Hlaváček, A., Obořilová, R., Skládal, P., Farka, Z., and Gorris, H.H.: Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay, Anal. Chem. 2023 95, 51, 4753 – 4759, https://doi.org/10.1021/acs.analchem.2c05670