Single‐reaction multi‐antigen serological test for comprehensive evaluation of SARS‐CoV‐2 patients by flow cytometry

  1. Yaiza Cáceres‐Martell
  2. Daniel Fernández‐Soto
  3. Carmen Campos‐Silva
  4. Eva M. García‐Cuesta
  5. Jose M Casasnovas
  6. David Navas‐Herrera
  7. Alexandra Beneítez‐Martínez
  8. Pedro Martínez‐Fleta
  9. Arantzazu Alfranca
  10. Francisco Sánchez‐Madrid
  11. Gabriela Escudero‐López
  12. Carlos Vilches
  13. Ricardo Jara‐Acevedo
  14. Hugh T. Reyburn
  15. José M. Rodríguez‐Frade
  16. Mar Valés‐Gómez

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Abstract

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen‐based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS‐CoV‐2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine‐like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory‐confirmed patients no SARS‐CoV‐2‐specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS‐CoV‐2 virus might provide insight into the wide array of clinical presentations of COVID‐19.

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  1. Version published to 10.1002/eji.202149319
  2. ScreenIT

    SciScore for 10.1101/2021.04.08.21254348: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Patient selection, samples and Institutional Review Board permits: Experiments were carried out following the ethical principles established in the Declaration of Helsinki. Patients (or their representatives) were informed about the study and gave a written informed consent.
    IACUC: Implications for therapeutic decision-making” approved by La Princesa Health Research Institute Research Ethics Committee (register # 4070) were used; samples and data from patients with severe vs mild disease were provided by the Biobank Hospital Universitario Puerta de Hierro Majadahonda (HUPHM)/Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana (IDIPHISA) (PT17/0015/0020 in the Spanish National Biobanks Network), they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees.
    RandomizationSamples were stratified and randomly spliced into a training and a test set.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Beads were incubated with either rabbit anti-His-tag antibody (Proteintech Group) or plasma from patients or healthy donors in a final volume of 50 μl in 96-well-plates (Nunc™ MicroWell™ 96-Well, Thermo Fisher Scientific) using the dilutions indicated in each experiment.
    anti-His-tag
    suggested: None
    To visualize antibody bound to antigen-coated beads, either PE-conjugated anti-rabbit antibody (0.25 μg/ml, Southern Biotech)
    PE-conjugated anti-rabbit antibody ( 0.25 μg/ml , Southern Biotech)
    suggested: None
    anti-rabbit
    suggested: None
    , PE-conjugated anti-human IgG and IgM, or FITC-conjugated anti-human IgA antibody (Immunostep S.L.) were added (30 μL/well) and incubated for 20 minutes at room temperature under agitation.
    anti-human IgG
    suggested: None
    IgM
    suggested: None
    anti-human IgA
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant cDNAs coding for soluble S (residues 1 to 1208) and RBD (332 to 534) proteins were cloned in the pcDNA3.1 vector for expression in HEK-293F cells using standard transfection methods.
    HEK-293F
    suggested: RRID:CVCL_6642)
    Software and Algorithms
    SentencesResources
    Statistical analysis: To assess the prediction capacity of the new methodology, an algorithm was built using Scikit-learn python package [11] (code available on request).
    Scikit-learn
    suggested: (scikit-learn, RRID:SCR_002577)
    python
    suggested: (IPython, RRID:SCR_001658)
    Comparison between severe and mild patients in each variable was performed by multiple t-tests followed by False Discovery Rate (1%) correction by two-stage step-up method in Graph Pad Prism 8 Software (GraphPad Software, USA, www.graphpad.com).
    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:
    In addition to an impact on early classification of patients, current limitations in the availability of vaccine doses suggest a novel possible application for sensitive multi-antigen assays for SARS-CoV-2 seropositivity. It has been shown that the antibody response to the first vaccine dose in individuals with pre-existing immunity is comparable or greater to that observed in naïve individuals who have been immunized twice [17]. Screening of the unvaccinated population with an assay sufficiently sensitive to identify individuals previously infected despite waning of antibody titres over time, would allow these individuals to be given the vaccine as a single booster, sparing them from possible suffering and complications after a second dose, and freeing up many urgently needed vaccine doses to be given to individuals with no protection. The test described here would also provide comprehensive information to support selection of convalescent sera or plasma for therapeutic use. Our data indicate the importance of this multi-antigen, multi-isotype analysis to detect potential SARS-CoV-2 reinfections in vaccinated individuals and suggest a possible use in establishing alternative vaccine administration routes that may elicit more potent IgA responses. This multi-antigen and multi-Ig assay can be easily modified for detection of antibodies in other fluids as saliva and breast milk. It is also highly tunable to different research needs, including detection of different immunoglobul...

    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.

  3. Version published to 10.1101/2021.04.08.21254348 on medRxiv