A Multiplex Microsphere IgG Assay for SARS-CoV-2 Using ACE2-Mediated Inhibition as a Surrogate for Neutralization

  1. Andrew Cameron
  2. Claire A. Porterfield
  3. Larry D. Byron
  4. Jiong Wang
  5. Zachary Pearson
  6. Jessica L. Bohrhunter
  7. Anthony B. Cardillo
  8. Lindsay Ryan-Muntz
  9. Ryan A. Sorensen
  10. Mary T. Caserta
  11. Stephen Angeloni
  12. Dwight J. Hardy
  13. Martin S. Zand
  14. Nicole D. Pecora

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Abstract

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens—spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP).

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  1. ScreenIT

    SciScore for 10.1101/2020.10.05.20203976: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was approved by the University of Rochester Institutional Review Board (RSRB STUDY00004836).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Study design and analysis: Statistical and graphical analysis: Statistical calculations and plotting were performed in Prism 8 (GraphPad Software, San Diego, CA).
    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: We detected the following sentences addressing limitations in the study:
    A limitation of this study is its dependence on retrospective and remnant serum samples which hindered longitudinal sampling. However, for a subset of nine individuals admitted to the ICU, we had multiple samples that enabled us to track their antibody and viral load data over time. This allowed us to look beyond the composite data (in Figure 1B) for a depiction of individual responses (Figure 2). The longitudinal MFI values of these individual patients suggested a high degree of interperson variation in terms of both kinetics and relative antigen responses. For most of these nine patients the MFIs for anti-S antibodies were higher. This is despite the fact that S protein was coupled to beads at only 10% of the molarity of RBD or NP. Thus, anti-S antibodies potentially comprise a larger proportion of anti-SARS-CoV-2 antibodies. We observed interperson and time (from symptom onset)-dependent variability in the ability of soluble ACE2 to block the binding of anti-S and anti-RBD antibodies, an indirect measure of neutralizing capability. In general, a trend towards greater neutralization over time from symptom onset was observed. The ability to measure inhibition of signal with ACE2 may prove useful to measure the effectiveness of, and discriminate between, the immune responses of infected or vaccinated individuals. However, an important caveat is that it is unclear how in vitro ‘neutralization’ performed in this manner translates to in vivo immunity. In summary, the 3Flex assay...

    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 10.1128/jcm.02489-20
  3. Version published to 10.1101/2020.10.05.20203976v1 on medRxiv