Serological Profile Of Specific Antibodies Against Dominant Antigens Of SARS-CoV-2 In Chilean COVID-19 Patients.

  1. K. Cereceda
  2. R. González-Stegmaier
  3. JL. Briones
  4. C. Selman
  5. A. Aguirre
  6. G. Valenzuela-Nieto
  7. C. Caglevic
  8. R. Gazitua
  9. A. Rojas-Fernandez
  10. F. Villarroel-Espíndola

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Abstract

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has been a pandemic since March 2020. Currently, the virus has infected more than 50 million people worldwide and more than half a million in Chile. For many coronaviruses, Spike (S) and Nucleocapsid (N) proteins are described as major antigenic molecules, inducing seroconversion and production of neutralizing antibodies. In this work, we evaluated the presence in serum of IgM, IgA and IgG antibodies against N and S proteins of SARS-CoV-2 using western blot, and developed an ELISA test for the qualitative characterization of COVID-19 patients. Patients with an active infection or who have recovered from COVID-19 showed specific immunoblotting patterns for the recombinants S protein and its domains S1 and S2, as well as for the N protein of SARS-CoV-2. Anti-N antibodies were more frequently detected than anti-S or anti-S1-RBD antibodies. People who were never exposed to SARS-CoV-2 did not show reactivity. Finally, indirect ELISA assays using N and S1-RBD proteins, alone or in combination, were established with variable sensitivity and specificity depending on the antigen bound to the solid phase. Overall, Spike showed higher specificity than the nucleocapsid, and comparable sensitivity for both antigens. Both approaches confirmed the seroconversion after infection and allowed us to implement the analysis of antibodies in blood for research purposes in a local facility.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.05.429566: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Recovered patients and those with an active infection with SARS-CoV-2 were invited to participate and signed a consent letter at the moment of enrollment to the clinical trial NCT04384588.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableInclusion criteria for convalescent patient considered men and women previously confirmed with COVID-19 by PCR test and 21 or more days after symptoms had finished.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Recombinant proteins and secondary antibodies: Recombinant SARS-CoV-2 Nucleocapsid Protein with C-terminal His-tag (
    His-tag
    suggested: None
    For the immunodetection, the membranes were separately incubated with either HRP-conjugated secondary antibody anti-human IgG, anti-human IgA or anti-human IgM diluted at 1:20,000 (0.04 μg/ml) in blocking solution.
    anti-human IgG
    suggested: None
    anti-human IgA
    suggested: None
    anti-human IgM
    suggested: None
    Later, each well was incubated at 20°C (±1°C) for 1h with 100 μL of a 20 ng/ml solution of the specific peroxidase-conjugated affiniPure anti-Human isotype antibody, anti-IgG (Fcβ fragment specific) (#709-035-098, Jackson Immuno-Research), anti-IgA (Frα fragment specific) (#109-035-011, Jackson Immuno-Research) and anti-IgM (μ chain) (#709-035-073, Jackson Immuno-Research).
    anti-Human isotype antibody,
    suggested: None
    anti-IgG
    suggested: None
    anti-IgA
    suggested: None
    anti-IgM (μ chain)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Immunofluorescence: HeLa cells (ATCC® CCL-2) were transient transfected with a mammalian expression vector expressing a Spike-GFPSpark tag codon optimized fusion SinoBiological VG40589-ACGLN in 10cm plates, 24 h after transfection ~ 8000 cells per well were deposited into 96 well optical plate (Themofisher), after 24 h incubation cells were washed with PBS (phosphate buffered saline solution) 1X 3 times and fixed with 4% paraformaldehyde at room temperature for 30 min.
    HeLa
    suggested: None
    Software and Algorithms
    SentencesResources
    Pixels were quantified using Fiji Software (v.2.0.0, NIH) and exposition time was used as reference.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    < Statistical analyses: Statistical analysis was performed using GraphPad Prism software, version 8.0.
    GraphPad Prism
    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:
    We understood the limitations of our work in the number of cases analyzed and the experimental strategy used; however, it provides information for some laboratories with limited resources to apply, exceptionally, basic research tools for monitoring COVID-19 cases during a pandemic.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04384588RecruitingCOVID19-Convalescent Plasma for Treating Patients With Activ…

    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 10.1101/2021.02.05.429566 on bioRxiv