Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations
Abstract
1. Introduction
2. Materials and Methods
2.1. Serum Collection
2.2. Recombinant Proteins Expressed in E. coli
2.3. Recombinant Proteins Expressed in HEK Cells
2.4. Peptide Synthesis
2.5. Enzyme-Linked Immunosorbent Assay (ELISA)
2.6. Peptide ELISA
3. Results
3.1. Expression and Purification of Recombinant SARS-CoV-2 Proteins
3.2. ELISA
3.3. RBD-Protein ELISA
3.4. S- and RBD-Peptide ELISA
3.5. M-Protein ELISA
3.6. M-Peptide ELISA
3.7. False Positive Results in N- and RBD-Protein ELISA
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Zhou, P.; Yang, X.-L.; Wang, X.-G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.-R.; Zhu, Y.; Li, B.; Huang, C.-L.; et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579, 270–273. [Google Scholar] [CrossRef]
- Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu, R.; et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 2020, 382, 727–733. [Google Scholar] [CrossRef] [PubMed]
- Johns Hopkins University & Medicine. COVID-19 Dashboard: By the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). Available online: https://coronavirus.jhu.edu/map.html (accessed on 22 May 2022).
- Chen, Y.; Liu, Q.; Guo, D. Emerging coronaviruses: Genome structure, replication, and pathogenesis. J. Med. Virol. 2020, 92, 2249. [Google Scholar] [CrossRef]
- Xu, Z.; Shi, L.; Wang, Y.; Zhang, J.; Huang, L.; Zhang, C.; Liu, S.; Zhao, P.; Liu, H.; Zhu, L.; et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir. Med. 2020, 8, 420–422. [Google Scholar] [CrossRef] [PubMed]
- Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T.S.; Herrler, G.; Wu, N.-H.; Nitsche, A.; et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181, 271–280.e8. [Google Scholar] [CrossRef] [PubMed]
- Letko, M.; Marzi, A.; Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 2020, 5, 562–569. [Google Scholar] [CrossRef] [PubMed]
- Zeng, W.; Liu, G.; Ma, H.; Zhao, D.; Yang, Y.; Liu, M.; Mohammed, A.; Zhao, C.; Yang, Y.; Xie, J.; et al. Biochemical characterization of SARS-CoV-2 nucleocapsid protein. Biochem. Biophys. Res. Commun. 2020, 527, 618–623. [Google Scholar] [CrossRef] [PubMed]
- Neuman, B.W.; Kiss, G.; Kunding, A.H.; Bhella, D.; Baksh, M.F.; Connelly, S.; Droese, B.; Klaus, J.P.; Makino, S.; Sawicki, S.G.; et al. A structural analysis of M protein in coronavirus assembly and morphology. J. Struct. Biol. 2011, 174, 11–22. [Google Scholar] [CrossRef]
- Nieto-Torres, J.L.; DeDiego, M.L.; Verdiá-Báguena, C.; Jimenez-Guardeño, J.M.; Regla-Nava, J.A.; Fernandez-Delgado, R.; Castaño-Rodriguez, C.; Alcaraz, A.; Torres, J.; Aguilella, V.M.; et al. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis. PLoS Pathog. 2014, 10, e1004077. [Google Scholar] [CrossRef] [PubMed]
- Hou, H.; Wang, T.; Zhang, B.; Luo, Y.; Mao, L.; Wang, F.; Wu, S.; Sun, Z. Detection of IgM and IgG antibodies in patients with coronavirus disease 2019. Clin. Transl. Immunol. 2020, 9, e01136. [Google Scholar] [CrossRef]
- Walls, A.C.; Park, Y.-J.; Tortorici, M.A.; Wall, A.; McGuire, A.T.; Veesler, D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 2020, 181, 281–292.e6. [Google Scholar] [CrossRef] [PubMed]
- Liu, D.; Wu, F.; Cen, Y.; Ye, L.; Shi, X.; Huang, Y.; Fang, S.; Ma, L. Comparative research on nucleocapsid and spike glycoprotein as the rapid immunodetection targets of COVID-19 and establishment of immunoassay strips. Mol. Immunol. 2021, 131, 6–12. [Google Scholar] [CrossRef] [PubMed]
- Luo, J.; Klett, J.; Gabert, J.; Lipp, T.; Karbach, J.; Jäger, E.; Borte, S.; Hoffmann, R.; Milkovska-Stamenova, S. A Quantitative ELISA to Detect Anti-SARS-CoV-2 Spike IgG Antibodies in Infected Patients and Vaccinated Individuals. Microorganisms 2022, 10, 1812. [Google Scholar] [CrossRef]
- Eberhardt, K.A.; Dewald, F.; Heger, E.; Gieselmann, L.; Vanshylla, K.; Wirtz, M.; Kleipass, F.; Johannis, W.; Schommers, P.; Gruell, H.; et al. Evaluation of a New Spike (S)-Protein-Based Commercial Immunoassay for the Detection of Anti-SARS-CoV-2 IgG. Microorganisms 2021, 9, 733. [Google Scholar] [CrossRef] [PubMed]
- Algaissi, A.; Alfaleh, M.A.; Hala, S.; Abujamel, T.S.; Alamri, S.S.; Almahboub, S.A.; Alluhaybi, K.A.; Hobani, H.I.; Alsulaiman, R.M.; AlHarbi, R.H.; et al. SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients. Sci. Rep. 2020, 10, 16561. [Google Scholar] [CrossRef]
- Luo, J.; Brakel, A.; Krizsan, A.; Ludwig, T.; Mötzing, M.; Volke, D.; Lakowa, N.; Grünewald, T.; Lehmann, C.; Wolf, J.; et al. Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections. Virol. J. 2022, 19, 50. [Google Scholar] [CrossRef]
- Engel, C.; Wirkner, K.; Zeynalova, S.; Baber, R.; Binder, H.; Ceglarek, U.; Enzenbach, C.; Fuchs, M.; Hagendorff, A.; Henger, S.; et al. Cohort Profile: The LIFE-Adult-Study. Int. J. Epidemiol. 2022. Online ahead of print. [Google Scholar] [CrossRef]
- Loeffler, M.; Engel, C.; Ahnert, P.; Alfermann, D.; Arelin, K.; Baber, R.; Beutner, F.; Binder, H.; Brähler, E.; Burkhardt, R.; et al. The LIFE-Adult-Study: Objectives and design of a population-based cohort study with 10,000 deeply phenotyped adults in Germany. BMC Public Health 2015, 15, 691. [Google Scholar] [CrossRef]
- System Biosciences. PiggyBac™ Transposon Vector System—User Manual: Cat. #PBxxx-1. Available online: http://www.systembio.com/wp/wp-content/uploads/Manual_PiggyBac_System.pdf (accessed on 16 May 2022).
- Schwarze, M.; Krizsan, A.; Brakel, A.; Pohl, F.; Volke, D.; Hoffmann, R. Cross-Reactivity of IgG Antibodies and Virus Neutralization in mRNA-Vaccinated People Against Wild-Type SARS-CoV-2 and the Five Most Common SARS-CoV-2 Variants of Concern. Front. Immunol. 2022, 13, 915034. [Google Scholar] [CrossRef]
- Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L.; et al. Crystal Structure of the 2019-nCoV Spike Receptor-Binding Domain Bound with the ACE2 Receptor. BioRxiv 2020. [Google Scholar]
- Yan, R.; Zhang, Y.; Li, Y.; Xia, L.; Guo, Y.; Zhou, Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 2020, 367, 1444–1448. [Google Scholar] [CrossRef]
- He, Y.; Qi, J.; Xiao, L.; Shen, L.; Yu, W.; Hu, T. Purification and characterization of the receptor-binding domain of SARS-CoV-2 spike protein from Escherichia coli. Eng. Life Sci. 2021, 21, 453–460. [Google Scholar] [CrossRef]
- Rodrigo, T.G.; Diego, P.; César, Á.; Esteban, V.P.; Carolina, M.; Silvina, C.; Benjamín, S.S.; Agustín, S.; Silvia, N.; Rossana, C.; et al. An “In-House” ELISA for SARS-CoV-2 RBD Uncovers Elevated Immune Response at Higher Altitudes. medRxiv 2021. [Google Scholar]
- Tomas-Grau, R.H.; Ploper, D.; Ávila, C.L.; Vera Pingitore, E.; Maldonado Galdeano, C.; Chaves, S.; Socias, S.B.; Stagnetto, A.; Navarro, S.A.; Chahla, R.E.; et al. Elevated Humoral Immune Response to SARS-CoV-2 at High Altitudes Revealed by an Anti-RBD "In-House" ELISA. Front. Med. 2021, 8, 720988. [Google Scholar] [CrossRef] [PubMed]
- de La Guardia, C.; Rangel, G.; Villarreal, A.; Goodridge, A.; Fernández, P.L.; Lleonart, R. Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama. PLoS ONE 2021, 16, e0257351. [Google Scholar] [CrossRef]
- Schmoldt, A.; Benthe, H.F.; Haberland, G. Digitoxin metabolism by rat liver microsomes. Biochem. Pharmacol. 1975, 24, 1639–1641. [Google Scholar] [CrossRef]
- Bates, T.A.; Weinstein, J.B.; Farley, S.; Leier, H.C.; Messer, W.B.; Tafesse, F.G. Cross-reactivity of SARS-CoV structural protein antibodies against SARS-CoV-2. Cell Rep. 2021, 34, 108737. [Google Scholar] [CrossRef]
- Jörrißen, P.; Schütz, P.; Weiand, M.; Vollenberg, R.; Schrempf, I.M.; Ochs, K.; Frömmel, C.; Tepasse, P.-R.; Schmidt, H.; Zibert, A. Antibody Response to SARS-CoV-2 Membrane Protein in Patients of the Acute and Convalescent Phase of COVID-19. Front. Immunol. 2021, 12, 679841. [Google Scholar] [CrossRef] [PubMed]
- Naqvi, A.A.T.; Fatima, K.; Mohammad, T.; Fatima, U.; Singh, I.K.; Singh, A.; Atif, S.M.; Hariprasad, G.; Hasan, G.M.; Hassan, M.I. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. Biochim. Biophys. Acta Mol. Basis Dis. 2020, 1866, 165878. [Google Scholar] [CrossRef]
- Raj, R. Analysis of non-structural proteins, NSPs of SARS-CoV-2 as targets for computational drug designing. Biochem. Biophys. Rep. 2021, 25, 100847. [Google Scholar] [CrossRef] [PubMed]
- Liu, J.; Sun, Y.; Qi, J.; Chu, F.; Wu, H.; Gao, F.; Li, T.; Yan, J.; Gao, G.F. The membrane protein of severe acute respiratory syndrome coronavirus acts as a dominant immunogen revealed by a clustering region of novel functionally and structurally defined cytotoxic T-lymphocyte epitopes. J. Infect. Dis. 2010, 202, 1171–1180. [Google Scholar] [CrossRef] [PubMed]
- Galipeau, Y.; Siragam, V.; Laroche, G.; Marion, E.; Greig, M.; McGuinty, M.; Booth, R.A.; Durocher, Y.; Cuperlovic-Culf, M.; Bennett, S.A.L.; et al. Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2. EBioMedicine 2021, 74, 103700. [Google Scholar] [CrossRef] [PubMed]
- Donofrio, G.; Franceschi, V.; Macchi, F.; Russo, L.; Rocci, A.; Marchica, V.; Costa, F.; Giuliani, N.; Ferrari, C.; Missale, G. A Simplified SARS-CoV-2 Pseudovirus Neutralization Assay. Vaccines 2021, 9, 389. [Google Scholar] [CrossRef]
- Lu, Y.; Wang, J.; Li, Q.; Hu, H.; Lu, J.; Chen, Z. Advances in Neutralization Assays for SARS-CoV-2. Scand. J. Immunol. 2021, 94, 10. [Google Scholar] [CrossRef]
- Lopandić, Z.; Protić-Rosić, I.; Todorović, A.; Glamočlija, S.; Gnjatović, M.; Ćujic, D.; Gavrović-Jankulović, M. IgM and IgG Immunoreactivity of SARS-CoV-2 Recombinant M Protein. Int. J. Mol. Sci. 2021, 22, 4951. [Google Scholar] [CrossRef]
- Voss, D.; Pfefferle, S.; Drosten, C.; Stevermann, L.; Traggiai, E.; Lanzavecchia, A.; Becker, S. Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein. Virol. J. 2009, 6, 79. [Google Scholar] [CrossRef]
- Khan, A.A.; Alahmari, A.A.; Almuzaini, Y.; Alamri, F.; Alsofayan, Y.M.; Aburas, A.; Al-Muhsen, S.; van Kerkhove, M.; Yezli, S.; Ciottone, G.R.; et al. Potential Cross-Reactive Immunity to COVID-19 Infection in Individuals with Laboratory-Confirmed MERS-CoV Infection: A National Retrospective Cohort Study from Saudi Arabia. Front. Immunol. 2021, 12, 727989. [Google Scholar] [CrossRef]
ELISA | RBD | M | |
---|---|---|---|
IgG | Sensitivity | ||
[+] <7 d | 50.0% (9/18) | 33.3% (6/18) | |
[+] 7–14 d | 75.0% (9/12) | 41.7% (5/12) | |
[+] >14 d | 90.6% (77/85) | 41.2% (35/85) | |
Specificity | |||
SARS-CoV-2 [−] | 97.9% (92/94) | 88.5% (46/52) | |
IgA | Sensitivity | ||
[+] <7 d | 83.3% (15/18) | 50.0% (9/18) | |
[+] 7–14 d | 100.0% (12/12) | 66.7% (7/12) | |
[+] >14 d | 98.8% (82/83) | 31.8% (27/85) | |
Specificity | |||
SARS-CoV-2 [−] | 84.0% (79/94) | 88.5% (46/52) |
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Schwarze, M.; Luo, J.; Brakel, A.; Krizsan, A.; Lakowa, N.; Grünewald, T.; Lehmann, C.; Wolf, J.; Borte, S.; Milkovska-Stamenova, S.; et al. Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations. Pathogens 2022, 11, 1515. https://doi.org/10.3390/pathogens11121515
Schwarze M, Luo J, Brakel A, Krizsan A, Lakowa N, Grünewald T, Lehmann C, Wolf J, Borte S, Milkovska-Stamenova S, et al. Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations. Pathogens. 2022; 11(12):1515. https://doi.org/10.3390/pathogens11121515
Chicago/Turabian StyleSchwarze, Mandy, Ji Luo, Alexandra Brakel, Andor Krizsan, Nicole Lakowa, Thomas Grünewald, Claudia Lehmann, Johannes Wolf, Stephan Borte, Sanja Milkovska-Stamenova, and et al. 2022. "Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations" Pathogens 11, no. 12: 1515. https://doi.org/10.3390/pathogens11121515
APA StyleSchwarze, M., Luo, J., Brakel, A., Krizsan, A., Lakowa, N., Grünewald, T., Lehmann, C., Wolf, J., Borte, S., Milkovska-Stamenova, S., Gabert, J., Scholz, M., & Hoffmann, R. (2022). Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations. Pathogens, 11(12), 1515. https://doi.org/10.3390/pathogens11121515