A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection—SARS-CoV-2 as a Proof of Concept

  1. Juuso Rusanen
  2. Lauri Kareinen
  3. Leonora Szirovicza
  4. Hasan Uğurlu
  5. Lev Levanov
  6. Anu Jääskeläinen
  7. Maarit Ahava
  8. Satu Kurkela
  9. Kalle Saksela
  10. Klaus Hedman
  11. Olli Vapalahti
  12. Jussi Hepojoki

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Abstract

PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best.

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  1. Version published to 10.1128/mbio.00902-21
  2. ScreenIT

    SciScore for 10.1101/2020.12.07.20245167: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Patient data were collected and samples handled according to research permit approved by the local review board, permit HUS/32/2018 (Helsinki University Hospital, Finland)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The obtained clones (N=5) were analyzed for TMPRSS2 expression by immunoblotting with V5 antibody (Invitrogen).
    V5
    suggested: None
    Antigens and antibodies: The production and purification of SARS-CoV-2 NP and SP antigens, followed a described protocol14,19,20.
    Antigens
    suggested: None
    SP
    suggested: None
    Briefly, antibody mixes with equimolar concentrations of Eu- and AF-labeled anti-RBD and anti-NP antibody concentrations were prepared in RIPA buffer.
    anti-RBD
    suggested: None
    anti-NP
    suggested: None
    The signals produced by hCoV-229E and hCoV-NL63 were evaluated by mixing 10 µl (undiluted, 1:10, 1:25, 1:50, and 1:100 diluted in RIPA) of the cell culture supernatant with 10 µl of the antibody mixes (at Eu- and AF-labeled anti-NP/-RBD concentrations of 6 + 6 nM).
    anti-NP/-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Specifically, 1 ml of 0.22 µm filtered (Millipore) infectious supernatant of HEK293T cells transfected on a 10-cm plate 48 h earlier using 30 µg polyethylenimine with 5 µg pLenti6.3/V5-DEST TMPRSS2 (obtained from Biomedicum Functional Genomics Unit, University of Helsinki), and 5 µg p8.9NDSB17 plus 2-5 µg of pMD2.
    HEK293T
    suggested: None
    G (a gift from Didier Trono, Addgene plasmid #12259; https://n2t.net/addgene:12259; RRID:Addgene_12259) was added on Vero E6 cells seeded onto 6-well plates.
    Vero E6
    suggested: None
    Once confirmed p24 negative, a clonal population of Vero E6-TMPRSS2 cells was obtained by limiting dilution.
    Vero E6-TMPRSS2
    suggested: None
    The hCoV-NL63 stock was produced by inoculating human lung fibroblasts (MRC-5, from ATCC) with 500 µl of 1:100 diluted cell culture supernatants (approximately 1 × 106 TCID50/ml) for 1 h at 37°C 5% CO2.
    MRC-5
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Specifically, 1 ml of 0.22 µm filtered (Millipore) infectious supernatant of HEK293T cells transfected on a 10-cm plate 48 h earlier using 30 µg polyethylenimine with 5 µg pLenti6.3/V5-DEST TMPRSS2 (obtained from Biomedicum Functional Genomics Unit, University of Helsinki), and 5 µg p8.9NDSB17 plus 2-5 µg of pMD2.
    TMPRSS2
    suggested: None
    Recombinant DNA
    SentencesResources
    G (a gift from Didier Trono, Addgene plasmid #12259; https://n2t.net/addgene:12259; RRID:Addgene_12259) was added on Vero E6 cells seeded onto 6-well plates.
    detected: RRID:Addgene_12259)

    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 10.1101/2020.12.07.20245167 on medRxiv