SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

  1. Mikail Dogan
  2. Lina Kozhaya
  3. Lindsey Placek
  4. Courtney Gunter
  5. Mesut Yigit
  6. Rachel Hardy
  7. Matthew Plassmeyer
  8. Paige Coatney
  9. Kimberleigh Lillard
  10. Zaheer Bukhari
  11. Michael Kleinberg
  12. Chelsea Hayes
  13. Moshe Arditi
  14. Ellen Klapper
  15. Noah Merin
  16. Bruce Tsan-Tang Liang
  17. Raavi Gupta
  18. Oral Alpan
  19. Derya Unutmaz

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Abstract

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects ( n  = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.07.20148106: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consent was obtained from all participants in this study and was approved by the following IRBs: 1) IRB# SUNY:269846.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cells were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS; Atlanta Biologicals, Lawrenceville, GA), 8% GlutaMAX (Life Technologies), 8% sodium pyruvate, 8% MEM vitamins, 8% MEM nonessential amino acid, and 1% penicillin/streptomycin (all from Corning Cellgro) for 72 hours, collected using %0.05 Trypsin-0.53 mM EDTA (Corning Cellgro) and stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems) then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).
    anti-human IgG
    suggested: None
    WT and ACE2 over-expressing HEK-293T were also stained with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen).
    Mouse IgG2a
    suggested: None
    Anti-S-RBD antibody and ACE2-Fc were both tested at a 5 μ/mL starting concentration and in additional 5-fold serial dilutions.
    Anti-S-RBD
    suggested: None
    SARS-CoV-2 antibody detection using ELISA: To evaluate antibodies binding to CoV-2 S-RBD protein, SARS-CoV-2 Spike S1-RBD IgG and IgM ELISA Detection Kit from GenScript was used according to the manufacturer’s instructions.
    CoV-2 S-RBD protein, SARS-CoV-2 Spike S1-RBD IgG
    suggested: None
    Pseudotyped virus neutralization assay: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 (Fig 4c, d), GenScript clone ID 6D11F2, NAb#2 (Fig 4d) and GenScript clone ID 10G6H5, NAb#3 (Fig 4d), Invitrogen clone ID MA5-35939 Nab#4 (Fig 4d), recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 (Fig 4d) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37°C degrees.
    anti-SARS-CoV-2
    suggested: (Thermo Fisher Scientific Cat# MA5-35939, RRID:AB_2866551)
    human IgG1
    suggested: (Thermo Fisher Scientific Cat# MA5-35939, RRID:AB_2866551)
    The half-maximal inhibitory concentration for plasma (NT50), ACE2-Fc or monoclonal antibodies (IC50) was determined using 4-parameter nonlinear regression (GraphPad Prism 8.0).
    ACE2-Fc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To determine the virus titers, HEK-293T cells were transduced with full length ACE2-IRES-GFP, ACE2-mKO2 fusion construct lentiviruses and analyzed via flow cytometry for their reporter gene expression 72 hours after infection.
    HEK-293T
    suggested: None
    Software and Algorithms
    SentencesResources
    UConn Healthcare workers who tested positive for the virus by PCR were recruited and samples banked for future testing.
    UConn Healthcare
    suggested: None
    Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (Tree Star).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Generating human ACE2 over-expressing cells: Wildtype ACE2 sequence was obtained from Ensembl Gene Browser (Transcript ID: ENST00000252519.8) and codon optimized with SnapGene by removing restriction enzyme recognition sites that are necessary for subsequent molecular cloning steps, preserving the amino acid sequence.
    Ensembl Gene Browser
    suggested: None
    SnapGene
    suggested: (SnapGene, RRID:SCR_015052)
    Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The half-maximal inhibitory concentration for plasma (NT50), ACE2-Fc or monoclonal antibodies (IC50) was determined using 4-parameter nonlinear regression (GraphPad Prism 8.0).
    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 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 10.1038/s42003-021-01649-6
  3. Version published to 10.1101/2020.07.07.20148106v3 on medRxiv
  4. Version published to 10.1101/2020.07.07.20148106v2 on medRxiv
  5. ScreenIT

    SciScore for 10.1101/2020.07.07.20148106: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementWritten informed consent obtained from all participants in this study and was approved by the following IRBs: 1 ) IRB# SUNY:269846 .RandomizationIn this regard , it is interesting to note that a bruton tyrosine kinase ( BTK ) inhibitor , that targets Fc-receptor signaling in macrophages , is being tested in a randomized clinical trial 32 .Blindingnot detected.Power Analysisnot detected.Sex as a biological variableSubdividing the subjects by sex did not reveal any statistical difference in IgG levels at any of the disease stages , although hospitalized females in the non-ICU setting had significantly lower antibody levels than ICU/deceased patients , whereas the difference in males was not significant ( Fig . 2f) .Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n=87) and were absent in the negative controls.
    CoV-2 Spike protein or Nucleocapsid protein specific IgG
    suggested: None
    Other isotype antibodies (IgA, IgG1-4) were also detected.
    IgA, IgG1-4
    suggested: None
    CoV-2 infection2-6 To predict protection against CoV-2, it is critical to understand the quantity, quality and duration of the antibody responses during different stages of COVID-19 and in the convalescent period.
    CoV-2
    suggested: (Abcam Cat# ab272504, AB_2847845)
    In this assay, we immobilized biotinylated CoV-2 Spike protein receptor binding domain (RBD) or the Nucleoprotein (N) on streptavidin beads, to detect specific antibodies from patient plasma (Fig.
    CoV-2 Spike protein receptor binding domain (RBD)
    suggested: None
    Different antibody isotypes were measured using anti-Ig (IgG, IgA, IgM) specific secondary antibodies conjugated to a fluorescent tag (Fig. 1a).
    anti-Ig ( IgG
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>IgA , IgM</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using either anti-SRBD antibody or soluble ACE2-Fc, we show very high sensitivity in detecting Spike protein binding, down to picogram ranges (Fig. 1b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-SRBD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Furthermore, Nucleocapsid protein-specific IgG levels and S-RBD specific IgA positively correlated with S-RBD IgG antibodies (Supplementary Fig. 1b, c).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>S-RBD IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Notably, IgG1 subclass antibody levels were comparable to total IgG levels whereas the other subtypes were relatively lower (Fig. 2b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgG1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To evaluate membrane expression of Spike protein, cells were stained with recombinant soluble ACE2-Fc fusion protein followed by a secondary staining with an anti-Fc antibody (Fig 3a).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-Fc</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 overexpression of ACE2-IRES-GFP or ACE2mKO2 was confirmed by staining with CoV-2 Spike-protein fused with mouse Fc (mFc) and antimFc secondary antibody (Supplementary Fig. 2a, b).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antimFc</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Of note, there was a significant negative correlation between the number of days and the IgG or IgA to S-RBD, anti-nucleocapsid IgG or the NT50 values ( !" = -0.67) (Fig. 6d), suggesting a potential decline in antibody titers over time.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-nucleocapsid IgG</b></div>
        <div>suggested: (Imported from the IEDB Cat# 3E9, <a href="https://scicrunch.org/resources/Any/search?q=AB_2848062">AB_2848062</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization of the virus by antibodies (NAbs) is one of the goals to achieve protection against CoV-218.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>CoV-218</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">However, another study showed IgA antibodies, but not IgG, increased in severe patients28.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgA</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Although it will be important to follow the same individual subject convalescent plasma over time to better assess this finding, our data point towards a relatively short-lived antibody response to COVID-19.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>COVID-19</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">WT and ACE2 over-expressing HEK-293T were also stained with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Mouse IgG2a</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-S-RBD antibody and ACE2-Fc was tested both at 5 µg/mL starting concentration and in additional 5-fold serial dilutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Anti-S-RBD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotype virus neutralization assay Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#3 (Fig 4D), Genscript clone ID 6D11F2, NAb#2 (Fig 4D) and Genscript clone ID 10G6H5, NAb#1 (Fig 4D)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-SARS-CoV-2</b></div>
        <div>suggested: (Abcam Cat# ab272854, <a href="https://scicrunch.org/resources/Any/search?q=AB_2847844">AB_2847844</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>human IgG1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Percent infection obtained was normalized samples derived from cells infected with CoV-2 or SARS pseudotyped virus in the absence of plasma, ACE2-Fc or monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ACE2-Fc</b></div>
        <div>suggested: None</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">In addition, we also developed SARS Spike protein pseudotyped lentivirus, which similarly infected 293-ACE2 cells at almost 100% efficiency at higher virus supernatant volumes (Fig. 3f)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293-ACE2</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_DR94">CVCL_DR94</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-293T cells (ATCC; mycoplasma-free low passage stock) were transfected with the expression plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol as previously described33.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK-293T</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">Generating human ACE2 over-expressing cells Wildtype ACE2 sequence was obtained from Ensembl Gene Browser (Transcript ID: ENST00000252519.8) and codon optimized with SnapGene by removing restriction enzyme recognition sites necessary for subsequent molecular cloning steps, preserving the amino acid sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Ensembl Gene Browser</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>SnapGene</b></div>
        <div>suggested: (SnapGene, <a href="https://scicrunch.org/resources/Any/search?q=SCR_015052">SCR_015052</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry data were analyzed using FlowJo (BD biosciences).</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">Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software)</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>
    
      <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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Author contributions M.D., L.K. and D.U. conceived, designed the experiments. M.D., L.K., L.P., M.Y. and R.H. carried out the experiments. B.T.L. designed the clinical research study on UConn Healthcare workers and M.K. recruited participants and executed clinical protocols. R.G. and O.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>UConn Healthcare</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr></table>

    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. Version published to 10.1101/2020.07.07.20148106v1 on medRxiv