Skip to main content
SpringerLink
Log in

One-Step Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein Using Screened Fv-Antibodies

  • Original Article
  • Published:
BioChip Journal Aims and scope Submit manuscript

Abstract

Fv-antibodies against the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were screened from an Fv-antibody library, and a one-step immunoassay was performed to detect SARS-CoV-2 using real viral samples. The Fv-antibody library was prepared using site-directed mutagenesis of the CDR3 region, which was composed of 11 amino acids. To screen the target Escherichia coli from the Fv-antibody library, the expressed probes [N-terminal domain (NTD) labeled with GFP and C-terminal domain (CTD) labeled with GFP] were reacted separately with the Fv-antibody library. After oligonucleotide sequencing, two clones for each probe were selected as the final clones. The screened Fv-antibodies with the binding affinity to NTD (or CTD) were expressed as soluble proteins, and the affinity constant (KD) was calculated to be 25.4 nM for NTD and 26.9 nM for CTD. The expressed Fv-antibodies were used for the one-step immunoassay based on switching-peptides, which were bound to the expressed Fv-antibodies. The one-step immunoassay based on Fv-antibodies could be used for the linear detection of SARS-CoV-2 NP, and the limit of detection (LOD) was estimated to be 9.6 nM (438 ng/mL) for Anti-NTD and 14.1 nM (639 ng/mL) for Anti-CTD. For the demonstration of one-step immunoassay for SARS-CoV-2, NATtrol™ SARS-CoV-2 real sample was used, and the LOD was estimated to be 29.7 copies/mL (Ct = 39.5) using Anti-NTD and 117.8 copies/mL (Ct = 38.0) using Anti-CTD. The measured LOD for the detection of SARS-CoV-2 using a one-step immunoassay based on the switching-peptide was considered feasible for the medical diagnosis of COVID-19. Finally, the interaction between the screened Fv-antibodies and SARS-CoV-2 NP was investigated using docking simulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The data is available on request to the corresponding author.

References

  1. Wuertz, K.M., Barkei, E.K., Chen, W.-H., Martinez, E.J., Lakhal-Naouar, I., Jagodzinski, L.L., Paquin-Proulx, D., Gromowski, G.D., Swafford, I., Ganesh, A.: A SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge. NPJ Vaccines 6, 1–11 (2021). https://doi.org/10.1038/s41541-021-00392-7

    Article  CAS  Google Scholar 

  2. Zhang, Q., Xiang, R., Huo, S., Zhou, Y., Jiang, S., Wang, Q., Yu, F.: Molecular mechanism of interaction between SARS-CoV-2 and host cells and interventional therapy. Signal Transduct. Target. Ther. 6, 233 (2021). https://doi.org/10.1038/s41392-021-00653-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kim, S., Lee, J.-H.: Current advances in paper-based biosensor technologies for rapid COVID-19 diagnosis. BioChip J. (2022). https://doi.org/10.1007/s13206-022-00078-9

    Article  PubMed  PubMed Central  Google Scholar 

  4. Jung, J., Sung, J.S., Bong, J.-H., Kim, T.-H., Kwon, S., Bae, H.E., Kang, M.-J., Jose, J., Lee, M., Shin, H.-J.: One-step immunoassay of SARS-CoV-2 using screened Fv-antibodies and switching peptides. Biosens. Bioelectron. (2023). https://doi.org/10.1016/j.bios.2023.115834

    Article  PubMed  PubMed Central  Google Scholar 

  5. Sun, C., Chen, L., Yang, J., Luo, C., Zhang, Y., Li, J., Yang, J., Zhang, J., Xie, L.: SARS-CoV-2 and SARS-CoV spike-RBD structure and receptor binding comparison and potential implications on neutralizing antibody and vaccine development. Biorxiv (2020). https://doi.org/10.1101/2020.02.16.951723

    Article  PubMed  PubMed Central  Google Scholar 

  6. Feng, W., Xiang, Y., Wu, L., Chen, Z., Li, Q., Chen, J., Guo, Y., Xia, D., Chen, N., Zhang, L.: Nucleocapsid protein of SARS-CoV-2 is a potential target for developing new generation of vaccine. J. Clin. Lab. Anal. 36, e24479 (2022). https://doi.org/10.1002/jcla.24479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schnurra, C., Reiners, N., Biemann, R., Kaiser, T., Trawinski, H., Jassoy, C.: Comparison of the diagnostic sensitivity of SARS-CoV-2 nucleoprotein and glycoprotein-based antibody tests. J. Clin. Virol. 129, 104544 (2020). https://doi.org/10.1016/j.jcv.2020.104544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Raïch-Regué, D., Muñoz-Basagoiti, J., Perez-Zsolt, D., Noguera-Julian, M., Pradenas, E., Riveira-Muñoz, E., Giménez, N., Carabaza, A., Giménez, F., Saludes, V.: Performance of SARS-CoV-2 antigen-detecting rapid diagnostic tests for Omicron and other variants of concern. Front. Microbiol. 13, 810576 (2022). https://doi.org/10.3389/fmicb.2022.810576

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ha, Y., Kim, I.: Recent developments in innovative magnetic nanoparticles-based immunoassays: from improvement of conventional immunoassays to diagnosis of COVID-19. BioChip J. 16, 351–365 (2022). https://doi.org/10.1007/s13206-022-00064-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xu, J.L., Davis, M.M.: Diversity in the CDR3 region of VH is sufficient for most antibody specificities. Immunity 13, 37–45 (2000). https://doi.org/10.1016/S1074-7613(00)00006-6

    Article  CAS  PubMed  Google Scholar 

  11. Jung, J., Bong, J.-H., Sung, J.S., Park, J.-H., Kim, T.-H., Kwon, S., Kang, M.-J., Jose, J., Pyun, J.-C.: Immunoaffinity biosensors for the detection of SARS-CoV-1 using screened Fv-antibodies from an autodisplayed Fv-antibody library. Biosens. Bioelectron. (2023). https://doi.org/10.1016/j.bios.2023.115439

    Article  PubMed  PubMed Central  Google Scholar 

  12. Xie, S., Wang, J., Yu, X., Peng, T., Yao, K., Wang, S., Liang, D., Ke, Y., Wang, Z., Jiang, H.: Site-directed mutations of anti-amantadine scFv antibody by molecular dynamics simulation: prediction and validation. J. Mol. Model. 26, 1–9 (2020). https://doi.org/10.1007/s00894-020-4286-y

    Article  CAS  Google Scholar 

  13. Sung, J.S., Bong, J.-H., Lee, S.J., Jung, J., Kang, M.-J., Lee, M., Shim, W.-B., Jose, J., Pyun, J.-C.: One-step immunoassay for food allergens based on screened mimotopes from autodisplayed FV-antibody library. Biosens. Bioelectron. 202, 113976 (2022). https://doi.org/10.1016/j.bios.2022.113976

    Article  CAS  PubMed  Google Scholar 

  14. Jung, J., Bong, J.-H., Sung, J.S., Lee, S.J., Lee, M., Kang, M.-J., Jose, J., Pyun, J.-C.: Fluorescein and rhodamine B-binding domains from autodisplayed Fv-antibody library. Bioconj. Chem. 32, 2213–2223 (2021). https://doi.org/10.1021/acs.bioconjchem.1c00376

    Article  CAS  Google Scholar 

  15. Jung, J., Bong, J.-H., Lee, S.J., Kim, M.-J., Sung, J.S., Lee, M., Kang, M.-J., Song, J., Jose, J., Pyun, J.-C.: Screening of Fv antibodies with specific binding activities to monosodium urate and calcium pyrophosphate dihydrate crystals for the diagnosis of gout and pseudogout. ACS Appl. Biomater. 4, 3388–3397 (2021). https://doi.org/10.1021/acsabm.0c01680

    Article  CAS  Google Scholar 

  16. Sung, J.S., Bong, J.-H., Yun, T.G., Han, Y., Park, Y., Jung, J., Lee, S.J., Kang, M.-J., Jose, J., Lee, M.: Antibody-mediated screening of peptide inhibitors for monoamine oxidase-B (MAO-B) from an autodisplayed FV library. Bioconj. Chem. 33, 1166–1178 (2022). https://doi.org/10.1021/acs.bioconjchem.2c00107

    Article  CAS  Google Scholar 

  17. Lee, S.J., Bong, J.-H., Jung, J., Sung, J.S., Kang, M.-J., Jose, J., Pyun, J.-C.: Screening of biotin-binding FV-antibodies from autodisplayed FV-library on E. coli outer membrane. Anal. Chim. Acta (2021). https://doi.org/10.1016/j.aca.2021.338627

    Article  PubMed  PubMed Central  Google Scholar 

  18. Matsuo, T.: Viewing SARS-CoV-2 nucleocapsid protein in terms of molecular flexibility. Biology 10, 454 (2021). https://doi.org/10.3390/biology10060454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bong, J.-H., Kim, H.-R., Jung, J., Park, J.-H., Sung, J.S., Lee, C.K., Choi, K.-H., Shin, S.-S., Kang, M.-J., Kim, H.O.: Switching-peptides for one-step immunoassay and its application to the diagnosis of human hepatitis B. Biosens. Bioelectron. 178, 112996 (2021). https://doi.org/10.1016/j.bios.2021.112996

    Article  CAS  PubMed  Google Scholar 

  20. Kim, T.-H., Bong, J.-H., Kim, H.-R., Shim, W.-B., Kang, M.-J., Pyun, J.-C.: One-step immunoassay based on switching peptides for analyzing ochratoxin A in wines. J. Anal. Sci. Technol. 13, 1–12 (2022). https://doi.org/10.1186/s40543-022-00352-3

    Article  CAS  Google Scholar 

  21. Lee, C.K., Jung, J., Kim, H.-R., Bong, J.-H., Kim, T.-H., Park, J.-H., Kwon, S., Kang, M.-J., Pyun, J.-C.: One-step immunoassay for the detection of food-poisoning related bacteria using a switching peptide. Analyst 147, 5363–5371 (2022). https://doi.org/10.1039/d2an00940d

    Article  CAS  PubMed  Google Scholar 

  22. Park, J.-H., Song, Z., Bong, J.-H., Kim, H.-R., Kim, M.-J., Choi, K.-H., Shin, S.-S., Kang, M.-J., Lee, D.Y., Pyun, J.-C.: Electrochemical one-step immunoassay based on switching peptides and pyrolyzed carbon electrodes. ACS Sens. 7, 215–224 (2022). https://doi.org/10.1021/acssensors.1c01998

    Article  CAS  PubMed  Google Scholar 

  23. Rabaan, A.A., Tirupathi, R., Sule, A.A., Aldali, J., Mutair, A.A., Alhumaid, S., Muzaheed, G.N., Koritala, T., Adhikari, R.: Viral dynamics and real-time RT-PCR Ct values correlation with disease severity in COVID-19. Diagnostics 11, 1091 (2021). https://doi.org/10.3390/diagnostics11061091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Engelmann, I., Alidjinou, E.K., Ogiez, J., Pagneux, Q., Miloudi, S., Benhalima, I., Ouafi, M., Sane, F., Hober, D., Roussel, A.: Preanalytical issues and cycle threshold values in SARS-CoV-2 real-time RT-PCR testing: should test results include these? ACS Omega 6, 6528–6536 (2021). https://doi.org/10.1021/acsomega.1c00166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kifaro, E.G., Kim, M.J., Jung, S., Noh, J.-Y., Song, C.-S., Misinzo, G., Kim, S.K.: Direct reverse transcription real-time PCR of viral RNA from saliva samples using hydrogel microparticles. BioChip J. 16, 409–421 (2022). https://doi.org/10.1007/s13206-022-00065-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Corman, V.M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D.K., Bleicker, T., Brünink, S., Schneider, J., Schmidt, M.L.: Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eur. Surveill. 25, 2000045 (2020). https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045

    Article  Google Scholar 

  27. Huang, W.E., Lim, B., Hsu, C.C., Xiong, D., Wu, W., Yu, Y., Jia, H., Wang, Y., Zeng, Y., Ji, M.: RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2. Microb. Biotechnol. 13, 950–961 (2020). https://doi.org/10.1111/1751-7915.13586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fozouni, P., Son, S., de León Derby, M.D., Knott, G.J., Gray, C.N., D’Ambrosio, M.V., Zhao, C., Switz, N.A., Kumar, G.R., Stephens, S.I.: Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell 184, 323–333 (2021). https://doi.org/10.1016/j.cell.2020.12.001

    Article  CAS  PubMed  Google Scholar 

  29. Zhao, H., Liu, F., Xie, W., Zhou, T.-C., OuYang, J., Jin, L., Li, H., Zhao, C.-Y., Zhang, L., Wei, J.: Ultrasensitive supersandwich-type electrochemical sensor for SARS-CoV-2 from the infected COVID-19 patients using a smartphone. Sens. Actuator B Chem. 327, 128899 (2021). https://doi.org/10.1016/j.snb.2020.128899

    Article  CAS  Google Scholar 

  30. Białobrzeska, W., Ficek, M., Dec, B., Osella, S., Trzaskowski, B., Jaramillo-Botero, A., Pierpaoli, M., Rycewicz, M., Dashkevich, Y., Łęga, T.: Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation. Biosens. Bioelectron. 209, 114222 (2022). https://doi.org/10.1016/j.bios.2022.114222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Peng, T., Dong, L., Feng, X., Yang, Y., Wang, X., Niu, C., Liang, Z., Qu, W., Zou, Q., Dai, X.: Relationship between SARS-CoV-2 nucleocapsid protein and N gene and its application in antigen testing kits evaluation. Talanta 258, 124462 (2023). https://doi.org/10.1016/j.talanta.2023.124462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Duell, J., Dittrich, M., Bedke, T., Mueller, T., Eisele, F., Rosenwald, A., Rasche, L., Hartmann, E., Dandekar, T., Einsele, H.: Frequency of regulatory T cells determines the outcome of the T-cell-engaging antibody blinatumomab in patients with B-precursor ALL. Leukemia 31, 2181–2190 (2017). https://doi.org/10.1038/leu.2017.41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu, M., Wang, B., Wang, F., Yang, Z., Gao, D., Zhang, C., Ma, L., Yu, X.: Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner. Appl. Microbiol. Biotechnol. 103, 6071–6079 (2019). https://doi.org/10.1007/s00253-019-09925-6

    Article  CAS  PubMed  Google Scholar 

  34. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T.C., Waldo, G.S.: Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006). https://doi.org/10.1038/nbt1172

    Article  CAS  PubMed  Google Scholar 

  35. Eberhardt, J., Santos-Martins, D., Tillack, A.F., Forli, S.: AutoDock Vina 1.2. 0: New docking methods, expanded force field, and python bindings. J. Chem. Inf. Model. 61, 3891–3898 (2021). https://doi.org/10.1021/acs.jcim.1c00203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wei, S., Suryawanshi, H., Djandji, A., Kohl, E., Morgan, S., Hod, E.A., Whittier, S., Roth, K., Yeh, R., Alejaldre, J.C.: Field-deployable, rapid diagnostic testing of saliva for SARS-CoV-2. Sci. Rep. 11, 1–9 (2021). https://doi.org/10.1038/s41598-021-84792-8

    Article  CAS  Google Scholar 

  37. Park, J.-H., Lee, G.-Y., Song, Z., Bong, J.-H., Chang, Y.W., Cho, S., Kang, M.-J., Pyun, J.-C.: Capacitive biosensor based on vertically paired electrodes for the detection of SARS-CoV-2. Biosens. Bioelectron. 202, 113975 (2022). https://doi.org/10.1016/j.bios.2022.113975

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Jung, J., Bong, J.-H., Kim, H.-R., Park, J.-H., Lee, C.K., Kang, M.-J., Kim, H.O., Pyun, J.-C.: Anti-SARS-CoV-2 nucleoprotein antibodies derived from pig serum with a controlled specificity. BioChip J. 15, 195–203 (2021). https://doi.org/10.1007/s13206-021-00019-y

    Article  CAS  Google Scholar 

  39. Jung, J., Bong, J.-H., Kim, T.-H., Sung, J.S., Lee, C., Kang, M.-J., Kim, H.O., Shin, H.-J., Pyun, J.-C.: Isolation of antibodies against the spike protein of SARS-CoV from pig serum for competitive immunoassay. BioChip J. 15, 396–405 (2021). https://doi.org/10.1007/s13206-021-00033-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bong, J.-H., Kim, T.-H., Jung, J., Lee, S.J., Sung, J.S., Lee, C.K., Kang, M.-J., Kim, H.O., Pyun, J.-C.: Competitive immunoassay of SARS-CoV-2 using pig sera-derived anti-SARS-CoV-2 antibodies. BioChip J. 15, 1–9 (2021). https://doi.org/10.1007/s13206-021-00011-6

    Article  CAS  Google Scholar 

  41. Bong, J.-H., Kim, T.-H., Jung, J., Lee, S.J., Sung, J.S., Lee, C.K., Kang, M.-J., Kim, H.O., Pyun, J.-C.: Pig sera-derived anti-SARS-CoV-2 antibodies in surface plasmon resonance biosensors. BioChip J. 14, 358–368 (2020). https://doi.org/10.1007/s13206-020-4404-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Korea Health Industry Development Institute (KHIDI) of Korea [HV22C0131] and the National Research Foundation (NRF) of Korea [NRF-2020R1A5A101913111, RS-2023-00209053, and NRF-2021R1A2C209370611], and Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) [122010022SB010].

Author information

Authors and Affiliations

  1. Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea

    Jaeyong Jung, Jeong Soo Sung, Tae-Hun Kim & Jae-Chul Pyun

  2. Korea Institute of Science and Technology (KIST), Seoul, 02456, Korea

    Min-Jung Kang

  3. Institute of Pharmaceutical and Medical Chemistry, University of Munster, 48149, Münster, Germany

    Joachim Jose

  4. College of Veterinary Medicine, Chungnam National University, Daejeon, 34134, Korea

    Hyun-Jin Shin

Authors
  1. Jaeyong Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeong Soo Sung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tae-Hun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Min-Jung Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joachim Jose
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hyun-Jin Shin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jae-Chul Pyun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jae-Chul Pyun.

Ethics declarations

Conflict of interest

The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 25 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jung, J., Sung, J.S., Kim, TH. et al. One-Step Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein Using Screened Fv-Antibodies. BioChip J (2024). https://doi.org/10.1007/s13206-024-00151-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13206-024-00151-5

Keywords

Search

Navigation