Diagnostic potential of new linear epitopes derived from the N-terminal domain of the SARS-CoV-2 Glycoprotein S
-
Sajjad Bahman
,
Safar Farajnia
,
Effat Alizadeh
,
Farzin Seirafi
,
Hojjatollah Nozad Charoudeh
,
Mohammad Kazem Hosseini
Abstract
Background and Objectives: The aim of this study was to assess the effectiveness of a new linear epitope from the N-terminal domain (NTD) of the SARS-CoV-2 S protein in the diagnosis of COVID-19.
Materials and Methods: Serum samples from patients were confirmed to have COVID-19 by means of RT-PCR. The linear epitope sequence of the NTD was amplified by RT-PCR, inserted into an expression vector, and produced in Escherichi coli (DE3) pLysS. Subsequently, the recombinant proteins were purified and refolded. The interaction between the purified protein and the antibodies in COVID-19 patient sera was evaluated using ELISA.
Results: Sequencing verified that the N-terminal linear epitope was successfully cloned into the PET-22b vector with a 6His-tag at the C-terminal end. The presence of a 25 kDa band on SDS-PAGE indicated the successful purification of the recombinant protein using Ni-NTA chromatography. The results of ELISA showed that the NTD linear epitope had strong sensitivity (88%) and specificity (96%) for identifying viral infection in COVID-19 patients' blood samples.
Conclusion: The findings of this study demonstrated that the NTD linear epitopes of the SARS-CoV-2 spike protein exhibit significant sensitivity and specificity for the diagnosis of COVID-19 infection using serological techniques. However, further evaluations involving larger sample sizes across diverse ethnic populations is essential.
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28. Amellal H, Assaid N, Charoute H, Akarid K, Maaroufi A, Ezzikouri S, et al. Kinetics of specific anti-SARS-CoV-2 IgM, IgA, and IgG responses during the first 12 months after SARS-CoV-2 infection: A prospective longitudinal study. PLoS One 2023; 18(7): e0288557.
29. Batra M, Tian R, Zhang C, Clarence E, Sacher CS, Miranda JN, et al. Role of IgG against N-protein of SARS-CoV2 in COVID19 clinical outcomes. Sci Rep 2021; 11: 3455.
30. Al-Mughales JA, Al-Mughales TJ, Saadah OI. Monitoring Specific IgM and IgG Production among severe COVID-19 Patients using Qualitative and Quantitative Immunodiagnostic Assays: A Retrospective cohort study. Front Immunol 2021; 12: 705441.
31. Zhai L, Wu L, Li F, Burnham RS, Pizarro JC, Xu B. A Rapid method for Refolding cell surface Receptors and Ligands. Sci Rep 2016; 6: 26482.
32. Allahyari M, Mohabati R, Babaie J, Amiri S, Siavashani ZJ, Zare M, et al. Production of in-vitro refolded and highly antigenic SAG1 for development of a sensitive and specific Toxoplasma IgG ELISA. J Immunol Methods 2015; 416: 157-166.
33. Roy V, Fischinger S, Atyeo C, Slein M, Loos C, Balazs A, et al. SARS-CoV-2-specific ELISA development. J Immunol Methods 2020; 484-485: 112832.
34. MacMullan MA, Ibrayeva A, Trettner K, Deming L, Das S, Tran F, et al. ELISA detection of SARS-CoV-2 antibodies in saliva. Sci Rep 2020; 10: 20818.
2. Liang Y, Wang ML, Chien CS, Yarmishyn AA, Yang YP, Lai WY, et al. Highlight of Immune Pathogenic Response and Hematopathologic effect in SARS-CoV, MERS-CoV, and SARS-Cov-2 infection. Front Immunol 2020; 11: 1022.
3. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020; 5: 536-544.
4. World Health Organization (2024). COVID-19 epidemiological update – 19 January 2024.
https://www.who.int/publications/m/item/covid-19-epidemiological-update---19-january-2024
5. Drexler JF, Helmer A, Kirberg H, Reber U, Panning M, Müller M, et al. Poor clinical sensitivity of rapid antigen test for influenza A pandemic (H1N1) 2009 virus. Emerg Infect Dis 2009; 15: 1662-1664.
6. Tang T, Bidon M, Jaimes JA, Whittaker GR, Daniel S. Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral Res 2020; 178: 104792.
7. Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun 2020; 11: 1620.
8. Belouzard S, Millet JK, Licitra BN, Whittaker GR. Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 2012; 4: 1011-1033.
9. Farajnia S, Khajenasiri N, Farajnia S, Seyrafi F, Bakhtiyari N. Performance of protein N linear epitopes in serodiagnosis of COVID-19 infection. Bioimpacts 2024; 15: 30232.
10. Scohy A, Anantharajah A, Bodéus M, Kabamba-Mukadi B, Verroken A, Rodriguez-Villalobos H. Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis. J Clin Virol 2020; 129: 104455.
11. Mak GC, Cheng PK, Lau SS, Wong KK, Lau CS, Lam ET, et al. Evaluation of rapid antigen test for detection of SARS-CoV-2 virus. J Clin Virol 2020; 129: 104500.
12. Mertens P, De Vos N, Martiny D, Jassoy C, Mirazimi A, Cuypers L, et al. Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context. Front Med (Lausanne) 2020; 7: 225.
13. Krüttgen A, Cornelissen CG, Dreher M, Hornef MW, Imöhl M, Kleines M. Comparison of the SARS-CoV-2 Rapid antigen test to the real star Sars-CoV-2 RT PCR kit. J Virol Methods 2021; 288: 114024.
14. Gupta A, Khurana S, Das R, Srigyan D, Singh A, Mittal A, et al. Rapid chromatographic immunoassay-based evaluation of COVID-19: A cross-sectional, diagnostic test accuracy study & its implications for COVID-19 management in India. Indian J Med Res 2021; 153: 126-131.
15. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 2020; 181: 281-292.e6.
16. Amanat F, Krammer F. SARS-CoV-2 Vaccines: Status Report. Immunity 2020; 52: 583-589.
17. Liu Z, Xiao X, Wei X, Li J, Yang J, Tan H, et al. Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS-CoV-2. J Med Virol 2020; 92: 595-601.
18. Breitling J, Aebi M. N-linked protein glycosylation in the endoplasmic reticulum. Cold Spring Harb Perspect Biol 2013; 5: a013359.
19. Braakman I, Hebert DN. Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol 2013; 5: a013201.
20. Broer R, Boson B, Spaan W, Cosset FL, Corver J. Important Role for the Transmembrane Domain of severe acute Respiratory Syndrome Coronavirus spike protein during Entry. J Virol 2006; 80: 1302-1310.
21. Chung CY, Majewska NI, Wang Q, Paul JT, Betenbaugh MJ. SnapShot: N-Glycosylation Processing Pathways across Kingdoms. Cell 2017; 171: 258-258.e1.
22. Hoffmann M, Kleine-Weber H, Pöhlmann S. A Multibasic Cleavage Site in the spike protein of SARS-CoV-2 Is Essential for infection of human Lung cells. Mol Cell 2020; 78: 779-784.e5.
23. Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG, Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antiviral Res 2020; 176: 104742.
24. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020; 367: 1260-1263.
25. Chi X, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 2020; 369: 650-655.
26. Pallesen J, Wang N, Corbett KS, Wrapp D, Kirchdoerfer RN, Turner HL, et al. Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A 2017; 114: E7348-E7357.
27. Ranjbar M, Asadi M, Nourigorji M, Sarkari B, Mostafavi-Pour Z, Zomorodian K, et al. Development of a recombinant nucleocapsid protein-based ELISA for the detection of IgM and IgG antibodies to SARS-CoV-2. Biotechnol Appl Biochem 2022; 69: 2592-2598.
28. Amellal H, Assaid N, Charoute H, Akarid K, Maaroufi A, Ezzikouri S, et al. Kinetics of specific anti-SARS-CoV-2 IgM, IgA, and IgG responses during the first 12 months after SARS-CoV-2 infection: A prospective longitudinal study. PLoS One 2023; 18(7): e0288557.
29. Batra M, Tian R, Zhang C, Clarence E, Sacher CS, Miranda JN, et al. Role of IgG against N-protein of SARS-CoV2 in COVID19 clinical outcomes. Sci Rep 2021; 11: 3455.
30. Al-Mughales JA, Al-Mughales TJ, Saadah OI. Monitoring Specific IgM and IgG Production among severe COVID-19 Patients using Qualitative and Quantitative Immunodiagnostic Assays: A Retrospective cohort study. Front Immunol 2021; 12: 705441.
31. Zhai L, Wu L, Li F, Burnham RS, Pizarro JC, Xu B. A Rapid method for Refolding cell surface Receptors and Ligands. Sci Rep 2016; 6: 26482.
32. Allahyari M, Mohabati R, Babaie J, Amiri S, Siavashani ZJ, Zare M, et al. Production of in-vitro refolded and highly antigenic SAG1 for development of a sensitive and specific Toxoplasma IgG ELISA. J Immunol Methods 2015; 416: 157-166.
33. Roy V, Fischinger S, Atyeo C, Slein M, Loos C, Balazs A, et al. SARS-CoV-2-specific ELISA development. J Immunol Methods 2020; 484-485: 112832.
34. MacMullan MA, Ibrayeva A, Trettner K, Deming L, Das S, Tran F, et al. ELISA detection of SARS-CoV-2 antibodies in saliva. Sci Rep 2020; 10: 20818.
| Files | ||
| Issue | Vol 17 No 3 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i3.18831 | |
| Keywords | ||
| COVID-19 Epitopes Enzyme-linked immunosorbent assay Protein purification Serologic diagnosis | ||
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How to Cite
1.
Bahman S, Farajnia S, Alizadeh E, Seirafi F, Nozad Charoudeh H, Hosseini MK. Diagnostic potential of new linear epitopes derived from the N-terminal domain of the SARS-CoV-2 Glycoprotein S. Iran J Microbiol. 2025;17(3):480-487.
