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

Abstract

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. 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. This finding also 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. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5–99.9) and 95.0% (83.0–99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.

Patent intelligence of RNA viruses: Implications for combating emerging and re-emerging RNA virus based infectious diseases

“…In addition, during the year 2020, there is an upward trend visible for these genes due to COVID-19 pandemic [31] . The S gene, M gene, N gene and E gene are major genes that give rise to their respective proteins which are essential for the structural and functional properties of RNA viruses [32] . The vaccines having S-protein may induce more immunogenicity and it may be useful for vaccine development as it has the ability to neutralize antibodies and invokes strong T cell response [33] .…”

Development and characterisation of cysteine-based gold electrodes for the electrochemical biosensing of the SARS-CoV-2 spike antigen

“…The necessity for a rapid, simple, cheap, highly sensitive and selective method for determining SARS-CoV-2 or its fragments persists. 6 Numerous methods for diagnosing COVID-19 have been reported, including real-time polymerase chain reaction (RT-PCR), [7][8][9][10][11] lateral flow assay (LFA), 4,13 lateral flow immunoassay (LFIA), [14][15][16][17][18] plasmonic sensors, 19 enzyme-linked immunosorbent assay (ELISA), 20 loop-mediated isothermal amplification (LAMP), 21,22 clustered regularly interspaced short palindromic repeats (CRISPR), 23,24 UV-visible spectroscopy 25 and electrochemical biosensing methods. [31][32][33][34][35][36][37][38][39][40][41][42][43][44] Among them, RT-PCR, despite being sensitive, selective, the most frequently used and arguably the most reliable method of diagnosing COVID-19, requires experienced personnel, and is expensive and time-consuming.…”

“…12 On the other hand, methods based on ELISA, LFA, LFIA and UV-visible spectroscopy, despite offering significant benefits such as being easy-to-use, cost-effective and moderately fast, have a low sensitivity that also returns false-negative results. 4,[13][14][15][16][17][18]20 Meanwhile, plasmonic sensor-, LAMP-and CRISPR-based methods 19,[21][22][23][24] are sensitive and cost-effective but involve complicated experimental processes and require experienced personnel.…”

Discriminative electrochemical biosensing of wildtype and omicron variant of SARS-CoV-2 nucleocapsid protein with single platform

“…RT-PCR has advantages involving being sensitive and selective, whereas it entails a long assay time and an expensive instrument [ [10] , [11] , [12] ]. Other commonly used diagnostic methods include lateral flow immunoassay (LFIA) [ [13] , [14] , [15] ], serological tests such as enzyme-linked immunosorbent assay (ELISA) [ 16 ], and electrochemical biosensors [ [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] ]. LFIA– and ELISA–based methods have mentioned benefits such as cheap, rapid and easy-to-use, whilst LFIA–based methods have lower sensitivity and ELISA–based methods are not proper for early diagnosis since they detect antibodies against viral antigens [ 16 , 26 ].…”

“…Other commonly used diagnostic methods include lateral flow immunoassay (LFIA) [ [13] , [14] , [15] ], serological tests such as enzyme-linked immunosorbent assay (ELISA) [ 16 ], and electrochemical biosensors [ [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] ]. LFIA– and ELISA–based methods have mentioned benefits such as cheap, rapid and easy-to-use, whilst LFIA–based methods have lower sensitivity and ELISA–based methods are not proper for early diagnosis since they detect antibodies against viral antigens [ 16 , 26 ]. By comparison, electrochemical methods have demonstrated many benefits involving ease of use, cost-effective and rapid analysis, high sensitivity and selectivity.…”