A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity

  1. Eric D. Laing
  2. Spencer L. Sterling
  3. Stephanie A. Richard
  4. Shreshta Phogat
  5. Emily C. Samuels
  6. Nusrat J. Epsi
  7. Lianying Yan
  8. Nicole Moreno
  9. Christian Coles
  10. Jennifer Mehalko
  11. Matthew Drew
  12. Caroline English
  13. Kevin K. Chung
  14. G. Travis Clifton
  15. Vincent J. Munster
  16. Emmie de Wit
  17. David Tribble
  18. Brian K. Agan
  19. Dominic Esposito
  20. Charlotte Lanteri
  21. Edward Mitre
  22. Timothy H. Burgess
  23. Christopher C. Broder

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Abstract

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 µL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.14.20207050: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: EPICC, IDCRP-085 and COVID-19 NYC protocols were approved by the Uniformed Services University Institutional Review Board.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Secondary antibody (goat anti-human IgG cross-absorbed biotin-conjugated or goat anti-human IgM cross-absorbed biotin-conjugated; Thermo Fisher Scientific, Waltham, MA) was diluted 1:5000 in 1XPBS + 0.05% Tween20 (PBST) and 100 µL of each secondary was added to each well and incubated for 45 minutes with agitation, and plates were washed three times.
    anti-human IgG
    suggested: (LSBio (LifeSpan Cat# LS-C22913-30, RRID:AB_899274)
    anti-human IgM
    suggested: (LSBio (LifeSpan Cat# LS-C5000-1000, RRID:AB_858362)
    As cross-reactive antibodies were found to occur in 4 out of 45 serum samples from archival HCoV PCR-positive individuals, we then established a cut-off of three standard deviations above the mean (99.7% probability) MFI of these archival HCoV convalescent serum samples (n= 43) to establish a positivity threshold for detection of SARS-CoV-2 spike protein reactive IgG and IgM antibodies.
    SARS-CoV-2 spike protein reactive IgG
    suggested: None
    IgM
    suggested: None
    One hundred microliters of secondary antibody, anti-human (H&L) HRP conjugated, diluted 1:5000 in PBS to each well was added to each well and plates were incubated at 37°C for 1 hour.
    anti-human (H&L
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A mock antigen, consisting of cell culture supernatant from untransfected HEK cells was collected via centrifugation then filtered through a 0.22 µM PES filter to remove debris.
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Figures were generated and statistical analyses were performed in GraphPad Prism version 7.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The positive predictive value and negative predictive value were calculated with MedCalc statistical software.
    MedCalc
    suggested: (MedCalc, RRID:SCR_015044)

    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 10.1101/2020.10.14.20207050v1 on medRxiv