Evaluation of recombinant nucleocapsid and spike proteins for serological diagnosis of novel coronavirus disease 2019 (COVID-19)

  1. Pingping Zhang
  2. Qi Gao
  3. Tang Wang
  4. Yuehua Ke
  5. Fei Mo
  6. Ruizhong Jia
  7. Wanbing Liu
  8. Lei Liu
  9. Shangen Zheng
  10. Yuzhen Liu
  11. Luping Li
  12. Yao Wang
  13. Lei Xu
  14. Kun Hao
  15. Ruifu Yang
  16. Shiyue Li
  17. Changqing Lin
  18. Yong Zhao

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Abstract

Background

The colloidal gold immunochromatography assay (GICA) is a rapid diagnostic tool for novel coronavirus disease 2019 (COVID-19) infections. However, with significant numbers of false negatives, improvements to GICA are needed.

Methods

Six recombinant HCoV-19 nucleocapsid and spike proteins were prepared and evaluated. The optimal proteins were employed to develop a sandwich-format GICA strip to detect total antibodies (IgM and IgG) against HCoV-19. GICA’s performance was assessed with comparison of viral RNA detection.

Results

Recombinant HCoV-19 proteins were obtained, including three prokaryotically expressed rN, rN1, rN2 nucleocapsid proteins, and three eukaryotically expressed rS1, rS-RBD, rS-RBD-mFc spike proteins. The recombinant proteins with the highest ELISA titers (rS1 and rS-RBD-mFc) against coronavirus-specific IgM and IgG were chosen for GICA development. The GICA has a sensitivity and specificity of 86.89% (106/122) and 99.39% (656/660), respectively. Furthermore, 65.63% (21/32) of the clinically confirmed but RT-PCR negative samples were GICA positive.

Conclusions

The eukaryotically-expressed spike proteins (rS1and rS-RBD-mFc) are more suitable than the prokaryotically expressed nucleocapsid proteins for HCoV-19 serological diagnosis. The GICA sandwich used to detect total antibodies is a powerful complement to the current standard RNA-based tests.

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  1. Version published to 10.1101/2020.03.17.20036954v2 on medRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.03.17.20036954: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics statement: This study was approved by the Medical Ethical Committee of Peking Union Medical College Hospital (approval number 002285), General Hosptical of Central Threater Command of the PLA (approval number 2020-003-1), and Shijiazhuang Fifth Hospital (approval number 2020-002).
    Consent: Written informed consent from all the patients was obtained, and all records and information on the patients were anonymized.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Using a dispenser (XYZ3000; BioDot, Irvine, CA), rS1 and the secondary polyclonal antibody (2 mg/mL) were coated onto the nitrocellulose membrane as the test and control lines, respectively, at a dispensing rate of 1.0 μL/cm.
    CA
    suggested: None
    rS1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Positive recombinant plasmids (210 μg each) and liposomes (280 μL) were diluted in 7 mL of opti-MEM medium for 5 min, respectively, mixed for 30 min and H293 cells (1.2× 106 /mL) were added.
    H293
    suggested: RRID:CVCL_H293)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.03.17.20036954v1 on medRxiv