Serological Test to Determine Exposure to SARS-CoV-2: ELISA Based on the Receptor-Binding Domain of the Spike Protein (S-RBDN318-V510) Expressed in Escherichia coli

  1. Alan Roberto Márquez-Ipiña
  2. Everardo González-González
  3. Iram Pablo Rodríguez-Sánchez
  4. Itzel Montserrat Lara-Mayorga
  5. Luis Alberto Mejía-Manzano
  6. Mónica Gabriela Sánchez-Salazar
  7. José Guillermo González-Valdez
  8. Rocio Ortiz-López
  9. Augusto Rojas-Martínez
  10. Grissel Trujillo-de Santiago
  11. Mario Moisés Alvarez

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Abstract

Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical. We report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor-binding domain of the SARS-CoV-2 spike protein (region of 193 amino acid residues from asparagine-318 to valine-510) of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag-mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S-RBDN318-V510 and a commercial full-length spike protein were compared in ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in ELISAs that used the full-spike antigen than the S-RBDN318-V510. Attachment of the antigens to the ELISA surface using a layer of anti-histidine antibodies gave equivalent resolution for both S-RBDN318-V510 and the full-length spike protein. Results demonstrate that ELISA-functional SARS-CoV-2 antigens can be produced in bacterial cultures, and that S-RBDN318-V510 may represent a cost-effective alternative to the use of structurally more complex antigens in serological COVID-19 testing.

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  1. Version published to 10.3390/diagnostics11020271
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    SciScore for 10.1101/2020.09.15.20195503: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Samples were collected from patients after obtaining informed and signed written consent and in complete observance of good clinical practices, the principles stated in the Helsinki Declaration, and applicable lab operating procedures at Hospital Alfa.
    IACUC: The experimental protocol was approved on May 20th, 2020 by a named institutional committee (Alfa Medical Center, Research Comitte; resolution AMCCI-TECCOVID-001).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISA assays: We developed and characterized two ELISA strategies for the evaluation of presence of specific anti-SARS-CoV-2 antibodies (Figure 3), as described in the Results and Discussion.
    anti-SARS-CoV-2
    suggested: None
    The presence of rabbit antibodies was revealed by adding donkey anti-rabbit-HRP (100 µL, 1:5000 dilution; Pierce
    anti-rabbit-HRP
    suggested: (Kindle Biosciences Cat# R1006, RRID:AB_2800464)
    Software and Algorithms
    SentencesResources
    Design of S-RBDN318-V510 and prediction of its 3D structure: We used the Geneious 11.1.5 software (Biomatters, Ltd., New Zealand) to design the vector pFH8-RBD SARS-COV2 that contained the RBD (region of 193 aa from N318-V510) of the SARS-CoV-2 spike protein.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    The 3D structure of the RBD protein with tags was predicted using the software I-TASSER server (University of Michigan, USA).
    I-TASSER
    suggested: (I-TASSER, RRID:SCR_014627)
    The degree of purity of S-RBDN318-V510 was estimated from SDS-PAGE protein profiles using Image J, an open source software for scanning densitometry analysis.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    The second ELISA format consisted of first sensitizing the plate wells with mouse anti-histidine pAb (100 µL, 1:1000 dilution; Bio-Rad Laboratories, Inc., CA, USA) and incubating for 8h at 4°C, then blocking with skim milk for 1 h at room temperature, followed by 3 washes with PBS containing 0.05% Tween-20™.
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 5. 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

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  3. Version published to 10.1101/2020.09.15.20195503 on medRxiv