Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

  1. Leire Egia-Mendikute
  2. Alexandre Bosch
  3. Endika Prieto-Fernández
  4. So Young Lee
  5. Borja Jiménez-Lasheras
  6. Ana García del Río
  7. Asier Antoñana-Vildosola
  8. Chiara Bruzzone
  9. Maider Bizkarguenaga
  10. Nieves Embade
  11. Rubén Gil-Redondo
  12. María Luz Martínez-Chantar
  13. Marcos López-Hoyos
  14. Nicola G. A. Abrescia
  15. José M. Mato
  16. Óscar Millet
  17. Asís Palazón

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Abstract

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.

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  1. Version published to 10.1038/s42003-021-02011-6
  2. Version published to 10.1101/2020.07.28.20162941v3 on medRxiv
  3. Version published to 10.1101/2020.07.28.20162941v2 on medRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.07.28.20162941: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Serum samples: Serum samples corresponding to preCOVID and acute COVID individuals were provided by the Basque Biobank (www.biobancovasco.org) after approval from the corresponding ethics committee (CEIC-E 20-26, 1-2016).
    Consent: All participants in the study provided informed consent and were anonymized.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Secondary antibodies were diluted in 100 µL of PBS containing 5% FBS: anti-human IgG-PE (1:50) (Clone G18-145, BD Biosciences Cat#555787
    anti-human IgG-PE
    suggested: (Santa Cruz Biotechnology Cat# sc-3756, RRID:AB_654591)
    ) and anti-human IgM-BV421 antibodies (1:1000) (Clone G20-127, BD Biosciences Cat#555783).
    anti-human IgM-BV421
    suggested: None
    After three washes with 250 μL of PBST in a plate washer (Biotek), each well was incubated with an anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (1:5000) (GenScript Cat#A01854) or anti-human IgM-HRP (Novus Biologicals Cat#NBP1-75014) for 1 hour at RT.
    anti-human IgG-horseradish
    suggested: None
    anti-human IgM-HRP
    suggested: (SouthernBiotech Cat# 2020-05, RRID:AB_2795603)
    Pearson correlation analyses between gMFI values obtained by C19BA and percentage of inhibition obtained by the neutralization assay for anti-RBD, anti-S1 and anti-N IgG antibodies were calculated using Prism 8 (GraphPad) considering a 95% confidence interval.
    anti-RBD
    suggested: None
    anti-S1
    suggested: None
    anti-N IgG
    suggested: None
    Software and Algorithms
    SentencesResources
    Results were analyzed using FlowJo version 10 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Neutralization assay: Binding of RBD to recombinant ACE2 was measured by a commercial surrogate virus neutralization test (sVNT) (cPass™ SARS-CoV-2
    SARS-CoV-2
    suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
    Data were analyzed using Prism 8 (GraphPad)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Phylogram generated from the FASTA alignment file was performed using FastTree (https://www.genome.jp/).
    FastTree
    suggested: (FastTree, RRID:SCR_015501)
    https://www.genome.jp/
    suggested: (GenomeNet, RRID:SCR_004165)

    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.

  5. ScreenIT

    SciScore for 10.1101/2020.07.28.20162941: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll serum samples were provided by the Basque Biobank (www.biobancovasco.org) after approval from the corresponding ethics committee (CEIC-E 20-26, 1-2016).Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISAs require optimisation and the use of individual plates for each antigen or antibody to be tested.
    antigen
    suggested: None
    A minority of COVID samples presented only N-reactive IgG and IgM antibodies, testing negative for RBD or S1.
    IgM
    suggested: None
    S1
    suggested: None
    Secondary antibodies were diluted in 100 µL of PBS containing 5% FBS: anti-human IgG-PE (1:50) (BD Biosciences #555787) and anti-human IgM-BV421 antibodies (1:1000) (BD Biosciences #555783).
    anti-human IgG-PE
    suggested: (Santa Cruz Biotechnology Cat# sc-3756, RRID:AB_654591)
    anti-human IgM-BV421
    suggested: None
    After three washes with 250 μL of PBST in a plate washer (Biotek), each well was incubated with an anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (1:5000) (GenScript #A01854) or antihuman IgM-HRP (Novus Biologicals #NBP1-75014) for 1 hour at RT.
    anti-human IgG-horseradish
    suggested: None
    antihuman IgM-HRP
    suggested: None
    Software and Algorithms
    SentencesResources
    PMMA (polymethyl methacrylate) 8.2 μm microbeads coated with streptavidin were purchased from PolyAn (#10652009).
    PolyAn
    suggested: None
    Results were analyzed using FlowJo version 10 (BD Biosciences). ELISA.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Data were analyzed using Prism 8 (GraphPad).
    GraphPad
    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 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.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  6. ScreenIT

    SciScore for 10.1101/2020.07.28.20162941: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll serum samples were provided by the Basque Biobank (www.biobancovasco.org) after approval from the corresponding ethics committee (CEIC-E 20-26, 1-2016).Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISAs require optimisation and the use of individual plates for each antigen or antibody to be tested.
    antigen
    suggested: None
    A minority of COVID samples presented only N-reactive IgG and IgM antibodies, testing negative for RBD or S1.
    IgM
    suggested: None
    S1
    suggested: None
    Secondary antibodies were diluted in 100 µL of PBS containing 5% FBS: anti-human IgG-PE (1:50) (BD Biosciences #555787) and anti-human IgM-BV421 antibodies (1:1000) (BD Biosciences #555783).
    anti-human IgG-PE
    suggested: (Santa Cruz Biotechnology Cat# sc-3756, RRID:AB_654591)
    anti-human IgM-BV421
    suggested: None
    After three washes with 250 μL of PBST in a plate washer (Biotek), each well was incubated with an anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (1:5000) (GenScript #A01854) or antihuman IgM-HRP (Novus Biologicals #NBP1-75014) for 1 hour at RT.
    anti-human IgG-horseradish
    suggested: None
    antihuman IgM-HRP
    suggested: None
    Software and Algorithms
    SentencesResources
    PMMA (polymethyl methacrylate) 8.2 μm microbeads coated with streptavidin were purchased from PolyAn (#10652009).
    PolyAn
    suggested: None
    Results were analyzed using FlowJo version 10 (BD Biosciences). ELISA.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Data were analyzed using Prism 8 (GraphPad).
    GraphPad
    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 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.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  7. ScreenIT

    SciScore for 10.1101/2020.07.28.20162941: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll serum samples were provided by the Basque Biobank (www.biobancovasco.org) after approval from the corresponding ethics committee (CEIC-E 20-26, 1-2016).Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISAs require optimisation and the use of individual plates for each antigen or antibody to be tested.
    antigen
    suggested: None
    The bead array (C19BA) is incubated with serum samples to allow the binding of anti-SARS-CoV-2 antibodies and then stained with anti-IgG and anti-IgM secondary antibodies labelled with different fluorochromes (Fig. 1a and Supplementary Fig.1).
    anti-IgG
    suggested: None
    Importantly, the sensitivity of C19BA was superior to ELISA (Fig. 1c) when their performance was compared in serial dilutions of recombinant anti-RBD and anti-N IgG antibodies.
    anti-RBD
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti-N IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Importantly, our assay identified the presence of N-reactive IgG antibodies in preCOVID samples, although in general at lower titers than in the COVID cohort (Fig. 2a,b). Fig. 2c shows representative dot plot profiles corresponding to preCOVID and COVID samples.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>N-reactive IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">While the reactivity against RBD and S1 was specific for COVID samples, cross-reactivity against N was observed in some preCOVID individuals that contained IgG, but not IgM antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A minority of COVID samples presented only N-reactive IgG and IgM antibodies, testing negative for RBD or S1.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In summary, we have developed a novel multiplexed method with higher sensitivity than traditional serology assays, using a triple combination of antigens that exploits the specificity of the Spike and RBD together with the less-specific N protein for the detection of antibodies against SARS-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SARS-CoV-2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibodies were diluted in 100 µL of PBS containing 5% FBS: anti-human IgG-PE (1:50) (BD Biosciences</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-human IgG-PE</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">#555787) and anti-human IgM-BV421 antibodies (1:1000) (BD Biosciences #555783).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-human IgM-BV421</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After three washes with 250 μL of PBST in a plate washer (Biotek), each well was incubated with an anti-human IgG-horseradish peroxidase (HRP) conjugated secondary antibody (1:5000) (GenScript #A01854) or antihuman IgM-HRP (Novus Biologicals #NBP1-75014) for 1 hour at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-human IgG-horseradish</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>antihuman IgM-HRP</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PMMA (polymethyl methacrylate) 8.2 μm microbeads coated with streptavidin were purchased from PolyAn (#10652009).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PolyAn</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Results were analyzed using FlowJo version 10 (BD Biosciences). ELISA.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>FlowJo</b></div>
        <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed using Prism 8 (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  8. Version published to 10.1101/2020.07.28.20162941v1 on medRxiv