Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2

  1. Dennis Lapuente
  2. Clara Maier
  3. Pascal Irrgang
  4. Julian Hübner
  5. Antonia Sophia Peter
  6. Markus Hoffmann
  7. Armin Ensser
  8. Katharina Ziegler
  9. Thomas H. Winkler
  10. Torsten Birkholz
  11. Andreas E. Kremer
  12. Philipp Steininger
  13. Klaus Korn
  14. Frank Neipel
  15. Klaus Überla
  16. Matthias Tenbusch

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Abstract

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid–based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections

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  1. Version published to 10.1007/s10096-020-04072-7
  2. ScreenIT

    SciScore for 10.1101/2020.05.09.20091447: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The implementation of the biobank has been approved by the local ethics committee of the UK Erlangen under the licence number AZ. 174_20 B.
    Consent: Five out of 60 were derived from plasma donors after (patients’ informed consents; approved by local ethics committee of the FAU; AZ. 2020, 49_20B).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Those specimens were not characterized in regard to anti-HCoV antibody status.
    anti-HCoV
    suggested: None
    100 μl FACS buffer was added, cells were centrifuged (500 xg, 4°C, 3min; used for all following centrifugation steps), washed two times with 180 μl FACS buffer, and bound antibodies were stained with secondary detection antibodies diluted 1:300 in 100 μl FACS buffer (30 min, 4°C, anti-IgG- AF647, clone HP6017, Biolegend, Cat #409320; anti-IgM-BV711, clone MHM-88, Biolegend, Cat #314540).
    anti-IgG-
    suggested: None
    anti-IgM-BV711
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    ) plasmids were used as marker proteins for transfected 293T cells.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Flow cytometric antibody assay: Human embryonic kidney cells (HEK 293T cells; ECACC 12022001) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Cat #11960-044) containing 10% fetal calf serum (Capricorn Scientific, Cat #FBS-12A), 1% GlutaMAX (Gibco, Cat #35050-038), and 1% Penicillin/Streptomycin (Gibco, Cat #15140-122) at 37°C and 5% CO2.
    HEK 293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were acquired on a BD LSRII or Thermo Fisher Attune Nxt cytometer and analysis was performed with FlowJo (Tree Star Inc.) or Flowlogic (Inivai Technologies).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

  3. Version published to 10.1101/2020.05.09.20091447 on medRxiv