Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19

Abstract

Diagnostics has played a significant role in effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays. Thus far, limited knowledge exists about the antigenic properties of the N protein. In this paper, we demonstrate the significant impact of dimerization of SARS-CoV-2 nucleocapsid protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics of COVID-19. The expressed purified protein from E.coli consists of two forms, dimeric and monomeric forms, which have been further characterized by biophysical and immunological means. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form of the protein. These findings have also been confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. It is evident that use of the dimeric form will increase the sensitivity of the current nucleocapsid dependent ELISA for rapid COVID-19 diagnostic. Further, the results indicate that monitoring and maintaining of the monomer-dimer composition is critical for accurate and robust diagnostics.

Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen

“…Given that SARS-CoV-2 serological methods have focused on S protein [13,20], a commercial SARS-CoV-2 full-length S protein-based IgG indirect ELISA was used as the second gold standard. As a monomer and an oligomer, the SARS-CoV-2 N protein has a molecular mass of 51.38 kDa and >670 kDa, respectively [32,33]. However, the SDS-PAGE silver-stained gel (Figure 1A) and the Western blot (Figure 1B) revealed a 47 kDa and 33 kDa band sizes of full-length (lane 2) and truncated (lane 3) N proteins, respectively.…”

Cell-free synthesis and purification of recombinant nucleocapsid (N), membrane (M), and envelope (E) proteins

“…There are at least 27 proteins in SARS-CoV-2. They include 15 non-structural proteins (nsp1~nsp10, nsp12~nsp16), four structural proteins (nucleocapsid, spike, membrane, and envelope proteins) and eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14) [5,6] . The 5'-end of the virus genome mainly encodes some of the non-structural proteins and the 3'-end encodes the structural proteins.…”

COVID-19 Pandemic and Vaccines Update on Challenges and Resolutions

“…In response to the outbreak, rapid diagnostics, speedy therapy, and vaccine research and development (R&D) are critical to curb the pandemic and prevent new viral outbreaks (Liu et al, 2020;Goel et al, 2021;Khan et al, 2021). Many attempts have been carried out on various platforms to develop a vaccine against SARS-CoV-2 (Callaway, 2020).…”