Ultra-Sensitive High-Resolution Profiling of Anti-SARS-CoV-2 Antibodies for Detecting Early Seroconversion in COVID-19 Patients

  1. Maia Norman
  2. Tal Gilboa
  3. Alana F. Ogata
  4. Adam M. Maley
  5. Limor Cohen
  6. Yongfei Cai
  7. Jun Zhang
  8. Jared E. Feldman
  9. Blake M. Hauser
  10. Timothy M. Caradonna
  11. Bing Chen
  12. Aaron G. Schmidt
  13. Galit Alter
  14. Richelle C. Charles
  15. Edward T. Ryan
  16. David R. Walt

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Abstract

The COVID-19 pandemic continues to infect millions of people worldwide. In order to curb its spread and reduce morbidity and mortality, it is essential to develop sensitive and quantitative methods that identify infected individuals and enable accurate population-wide screening of both past and present infection. Here we show that Single Molecule Array assays detect seroconversion in COVID-19 patients as soon as one day after symptom onset using less than a microliter of blood. This multiplexed assay format allows us to quantitate IgG, IgM and IgA immunoglobulins against four SARS-CoV-2 targets, thereby interrogating 12 antibody isotype-viral protein interactions to give a high resolution profile of the immune response. Using a cohort of samples collected prior to the outbreak as well as samples collected during the pandemic, we demonstrate a sensitivity of 86% and a specificity of 100% during the first week of infection, and 100% sensitivity and specificity thereafter. This assay should become the gold standard for COVID19 serological profiling and will be a valuable tool for answering important questions about the heterogeneity of clinical presentation seen in the ongoing pandemic.

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    SciScore for 10.1101/2020.04.28.20083691: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementAll samples were collected under approval of the Institutional Review Board for Huma Subjects Research at Massachusetts General Hospital.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Many serological enzyme-linked immunosorbent assays (ELISA) have been recently developed to detect anti-SARS-CoV-2 antibodies.
    anti-SARS-CoV-2
    suggested: None
    To address these limitations, we developed ultra-sensitive Single Molecule Array (Simoa) assays for anti-SARS-CoV-2 IgG, IgM, and IgA antibodies against four immunogenic viral proteins, providing us with detailed information about early stages of immune activation 17.
    IgA antibodies against four immunogenic viral proteins, providing us with detailed information about early stages of immune activation
    suggested: None
    Developing an ultra-sensitive Simoa assay for anti-SARS-CoV-2 antibodies We developed a multiplexed ultra-sensitive Simoa assay for detection of IgG, IgM, and IgA against SARS-CoV-2 in human plasma.
    IgA against SARS-CoV-2
    suggested: None
    Finally, the beads are incubated with the enzyme streptavidin-βgalactosidase (SβG), which binds to the biotinylated anti-human immunoglobulin antibody, forming a complete enzyme-labeled immunocomplex.
    anti-human immunoglobulin
    suggested: None
    We quantitatively validated viral target conjugation to the bead surface using anti-His tag antibodies as well as recombinant human anti-RBD antibody, as described in the Supplementary Information (Supplementary Figure 3).
    anti-His tag
    suggested: None
    For spike , S1 , and nucleocapsid , confirmation of antigen attachment to the beads was demonstrated by Simoa with His tags experiments using a biotinylated anti-His tag antibody ( ThermoFisher MA121315BTI ) on the HD-X Analyzer ( Quanterix) .
    S1
    suggested: None
    The anti-His-tag antibody was plated at concentrations of 0.1 pg/mL to 10,000 pg/mL using tenfold dilutions .
    anti-His-tag
    suggested: None
    RBD conjugation to beads was confirmed by Simoa with an anti-RBD antibody ( clone CR3022 ) and a biotinylated anti human-IgG antibody ( Bethyl Laboratories A80-148B)
    anti-RBD
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti human-IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Biotinylation Detection antibodies for IgA , IgG and IgM were purchased from Thermo Fisher , Bethyl Laboratories , Abcam , Biolegend , and R&D systems ( see Immunoglobulin Simoa assay format ) and were biotinylated for use in Simoa assays as described previously by Cohen et al 23 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgA , IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-human immunoglobulin antibodies were diluted in Homebrew Detector/Sample Diluent to final concentrations of: IgG (Bethyl Labratories A80-148B): 7.73ng/mL, IgM (Thermo Fisher MII0401): 216ng/mL, IgA (Abcam ab214003): 150ng/mL.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgM</b></div>
        <div>suggested: (Thermo Fisher Scientific Cat# MII0401, <a href="https://scicrunch.org/resources/Any/search?q=AB_11153935">AB_11153935</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</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">The expression construct was transiently transfected in HEK 293T cells using polyethylenimine.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK 293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></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">Logistic regression analysis was conducted in R version 3.6.2 for the multivariate analysis and Graphpad Prism 7 for the univariate analysis25.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Graphpad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All figures were plotted in Graphpad Prsim 7, Igor Pro7 and Adobe Illustrator version 2015. Acknowledgments: The authors would like to thank Liangxia Xie for the helpful discussion regarding the experimental design.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Graphpad</b></div>
        <div>suggested: (GraphPad, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000306">SCR_000306</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Adobe Illustrator</b></div>
        <div>suggested: (Adobe Illustrator, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010279">SCR_010279</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, support came from the Globa TravEpiNet (GTEN) system sponsored by the US Centers for Disease Control and Preventio (Grant No. U01CK000490: ETR, RCC) as well as a T32GM007753 grant from NIGMS, T32 AI007245 from NIAID, and an R01AI146779 from NIAID.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>NIAID</b></div>
        <div>suggested: (NIAID, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016598">SCR_016598</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">He is an inventor of the Simoa technology, a founder o the company and also serves on its Board of Directors. Dr. Walt’s interests were reviewed an are managed by BWH and Partners HealthCare in accordance with their conflict of interes policies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Partners HealthCare</b></div>
        <div>suggested: (Partners HealthCare Biobank, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001316">SCR_001316</a>)</div>
      </div>
    </td></tr></table>

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  2. Version published to 10.1101/2020.04.28.20083691v1 on medRxiv