24 People, one test: Boosting test efficiency using pooled serum antibody testing for SARS-CoV-2

  1. Stefan Nessler
  2. Jonas Franz
  3. Franziska van der Meer
  4. Konstantina Kolotourou
  5. Vivek Venkataramani
  6. Chalid Hasan
  7. Beatrix Pollok-Kopp
  8. Andreas E. Zautner
  9. Christine Stadelmann
  10. Michael Weig
  11. Stefan Pöhlmann
  12. Markus Hoffmann
  13. Joachim Riggert

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Abstract

Background

The global pandemic of COVID-19 (coronavirus disease 2019) is caused by the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), with different prevalence rates across countries and regions. Dynamic testing strategies are mandatory to establish efficient mitigation strategies against the disease; to be cost effective, they should adapt to regional prevalences. Seroprevalence surveys that detect individuals who have mounted an immune response against COVID-19 will help to determine the total number of infections within a community and improve the epidemiological calculations of attack and case fatality rates of the virus. They will also inform about the percentage of a population that might be immune against re-infections.

Methods

We developed a sensitive and specific cell-based assay to detect conformational SARS-CoV-2 spike (SARS-2-S) S1 antibodies in human serum, and have cross-evaluated this assay against two FDA-approved SARS-CoV-2 antibody assays. We performed pseudovirus neutralization assays to determine whether sera that were rated antibody-positive in our assay were able to specifically neutralize SARS-2-S. We pooled up to 24 sera and assessed the group testing performance of our cell-based assay. Group testing was further optimized by Monte Carlo like simulations and prospectively evaluated.

Findings

Highly significant correlations could be established between our cell-based assay and commercial antibody tests for SARS-CoV-2. SARS-2-S S1 antibody-positive sera neutralized SARS-2-S but not SARS-S, and were sensitively and specifically detected in pools of 24 samples. Monte Carlo like simulations demonstrated that a simple two-step pooling scheme with fixed pool sizes performed at least equally as well as Dorfman’s optimal testing across a wide range of antibody prevalences.

Interpretation

We demonstrate that a cell-based assay for SARS-2-S S1 antibodies qualifies for group testing of neutralizing anti-SARS-2-S antibodies. The assay can be combined with an easily implemented algorithm which greatly expands the screening capacity to detect anti-SARS-2-S antibodies across a wide range of antibody prevalences. It will thus improve population serological testing in many countries.

Funding

This work was supported by the Bundesministerium für Bildung und Forschung within the network project RAPID (risk assessment in pre-pandemic respiratory infectious diseases [grant number 01KI1723D, S.P.]).

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  1. ScreenIT

    SciScore for 10.1101/2020.09.01.20186130: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written consent was obtained from all individuals and the study was approved by the local ethics committee (14/8/20)
    IRB: Written consent was obtained from all individuals and the study was approved by the local ethics committee (14/8/20)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableThe female-to-male ratio was 1·77 and the median age was 42 years (
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Furthermore, we collected 589 blood donor samples from March to June 2020 to evaluate the potential of the cell-based assay to reliably detect anti-SARS-CoV-2 antibodies in pools of 21-24 patients.
    anti-SARS-CoV-2
    suggested: None
    Cell-based antibody measurements: HEK293 cells were stably transfected with either the pCMV3-2019-nCoV-Sl (HEKspike S1, Sino Biologicals VG40591-UT, China), pCMV3-HCoV-OC43-Spike-Flag (HEKOC43,
    pCMV3-HCoV-OC43-Spike-Flag
    suggested: None
    HEKOC43
    suggested: None
    HEKspike S1 cells with strong membrane staining by SARS-2-S S1 antibodies were enriched by cell sorting (BD FACSAria™ II) and cultivated further in DMEM, high glucose (Gibco™, Thermo Fisher Scientific, MA, USA) supplemented with 10% FCS (Gibco™), P/S (Gibco™) and 200
    S1
    suggested: None
    Individual human serum samples were pre-diluted 1 to 10 in DMEM growth medium and added to 20 000 HEKspike S1 or HEKEV cells in 96 U wells, yielding a final serum dilution of 1 to 50 to measure IgG or IgG1 anti-SARS-2-S spike S1 antibodies.
    anti-SARS-2-S spike S1
    suggested: None
    The following secondary antibodies were used: F(ab’)2 goat anti-human IgG-APC (1:100, Jackson ImmunoResearch, PA,
    anti-human IgG-APC
    suggested: None
    For the measurement of IgA or lgG3 anti-SARS-2-S S1 antibodies, human serum was diluted 1:25 or 1:5, respectively, to adjust for the reduced concentrations of these Ig isotypes in human serum.
    anti-SARS-2-S S1
    suggested: None
    The SARS-CoV-2-lgG immunoassay by Euroimmun is a commercially available ELISA (Euroimmun; Lübeck, Germany #EI 2606-9601 G), which detects anti-SARS-CoV-2 spike S1 antibodies with high diagnostic accuracy7 and was used in previous seroprevalence studies8.
    anti-SARS-CoV-2 spike S1
    suggested: None
    The following primary and secondary antibodies were used: anti-SARS-CoV-2 spike protein S1 receptor binding domain (1:250, mouse monoclonal #1034515, R&D systems, MN, USA); anti-β-actin antibody AC15 (1:5000, Sigma-Aldrich, Merck, Darmstadt, Germany) and polyclonal rabbit anti-mouse Ig/HRP (1:1000, DAKO, Jena, Germany).
    anti-SARS-CoV-2 spike protein S1 receptor binding domain
    suggested: None
    anti-β-actin
    suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
    anti-mouse Ig/HRP
    suggested: None
    Cells were washed three times with PBS and then incubated for 15 min with a F(ab’)2 goat anti-human lgG-AF647 antibody (1:100, Jackson ImmunoResearch, PA, USA) at 4 °C.
    anti-human lgG-AF647
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell-based antibody measurements: HEK293 cells were stably transfected with either the pCMV3-2019-nCoV-Sl (HEKspike S1, Sino Biologicals VG40591-UT, China), pCMV3-HCoV-OC43-Spike-Flag (HEKOC43,
    HEK293
    suggested: None
    In brief, replication-deficient vesicular stomatitis virus (VSV) pseudotype particles (VSVpp,10) bearing either VSV glycoprotein (VSV-G), SARS-S or SARS-2-S were incubated with four-fold serial dilutions of heat-inactivated (30 min at 56 °C) patient serum (final serum dilutions: 1:25, 1:100, 1:400, 1:1600, 1:6400) or medium without serum (control) for 30 min at 37 °C, before the mixtures were inoculated on Vero 76 cells (kindly provided by Andrea Maisner, Philipps University Marburg) in 96-well plates.
    Vero 76
    suggested: None
    Software and Algorithms
    SentencesResources
    The assay was run on the Abbott Architect instrument platform according to the manufacturer’s instructions.
    Abbott Architect
    suggested: (Abbott ARCHITECT i1000sr System, RRID:SCR_019328)
    Proteins were detected with Western Lightning® Plus ECL Enhanced Chemoluminescence Substrate (PerkinElmer) and the images were equally adjusted in Adobe Photoshop to enhance the visibility of the bands.
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

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  2. Version published to 10.1101/2020.09.01.20186130 on medRxiv