Automated Western immunoblotting detection of anti-SARS-CoV-2 serum antibodies

  1. Sophie Edouard
  2. Rita Jaafar
  3. Nicolas Orain
  4. Philippe Parola
  5. Philippe Colson
  6. Bernard La Scola
  7. Pierre-Edouard Fournier
  8. Didier Raoult
  9. Michel Drancourt

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Abstract

ELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary serologic assay for COVID-19. The Jess TM Simple Western system, an automated capillary-based assay, was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as the source of antigen, and total immunoglobulins (IgG, IgM, IgA) detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen’s Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2.

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  1. Version published to 10.1007/s10096-021-04203-8
  2. ScreenIT

    SciScore for 10.1101/2020.11.26.20238733: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: All sera were retrospectively tested and no sample was collected specifically for this study which was approved by our institution’s ethics committee under No.2020-024.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Of these, 16 serum samples were used for conventional immunoblotting including 3 samples exhibiting low (1:200), moderate (1:800) and high (1:3,200) IgG titers using IFA that were used to fix optimal conditions to be used for AWB (antigen, serum and secondary antibodies concentrations).
    antigen, serum
    suggested: None
    After a wash step, goat HRP-conjugated anti-human IgG/IgM/IgA antibodies (Jackson ImmunoResearch) diluted 1:500 was added for 30 min.
    anti-human IgG/IgM/IgA
    suggested: None
    Software and Algorithms
    SentencesResources
    For data comparisons and statistical analyses, the Fisher’s exact test, Chi-squared test, Mann-Witney test and standard statistical software (GraphPad Prism v7) were used.
    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: 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.

  3. Version published to 10.1101/2020.11.26.20238733 on medRxiv