Improved diagnosis of SARS-CoV-2 by using Nucleoprotein and Spike protein fragment 2 in quantitative dual ELISA tests

  1. Carolina De M. Verissimo
  2. Carol O’Brien
  3. Jesús López Corrales
  4. Amber Dorey
  5. Krystyna Cwiklinski
  6. Richard Lalor
  7. Jack M. Doyle
  8. Stephen Field
  9. Claire Masterson
  10. Eduardo Ribes Martinez
  11. Gerry Hughes
  12. Colm Bergin
  13. Kieran Walshe
  14. Bairbre McNicholas
  15. John G. Laffey
  16. John P. Dalton
  17. Colm Kerr
  18. Sean Doyle

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The novel Coronavirus, SARS-CoV-2, is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and Spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative ELISA assays that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive COVID-19 cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients that had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.04.07.21255024: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: James’s Hospital, Trinity College Dublin with informed consent.
    IRB: Human experimental work was conducted according to Human Research Ethics Committees.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableThe group consisted of 29 females and 13 males, ranging from 27 to 64 years old (average 41.5).

    Table 2: Resources

    Antibodies
    SentencesResources
    To further confirm the expression and purification of the recombinant proteins, Western blots were performed using a monoclonal mouse anti-polyhistidine antibody (diluted 1:5,000) (Sigma-Aldrich) as a primary antibody followed by incubation with a secondary antibody alkaline phosphatase or horseradish peroxidase (HRP)-conjugated goat to mouse-anti-IgG (diluted 1:5000) (Sigma-Aldrich).
    anti-polyhistidine
    suggested: None
    mouse-anti-IgG
    suggested: None
    After washing five times with PBST, the secondary antibody HRP goat anti-human IgG (Fc specific) (Sigma-Aldrich) was added (1:15,000), and the plates were incubated for 1 hr at 37°C.
    anti-human IgG
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analysed using Prism 5 (Graphpad).
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    Statistical analyses: Statistical analysis was carried out using GraphPad Prism version 5.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 did not find any issues relating to the usage of bar graphs.

    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.

  2. Version published to 10.1101/2021.04.07.21255024 on medRxiv
  3. Version published to 10.1017/s0950268821001308