SARS-CoV-2 Nucleocapsid Protein TR-FRET Assay Amenable to High Throughput Screening

  1. Kirill Gorshkov
  2. Desarey Morales Vasquez
  3. Kevin Chiem
  4. Chengjin Ye
  5. Bruce Nguyen Tran
  6. Juan Carlos de la Torre
  7. Thomas Moran
  8. Catherine Z. Chen
  9. Luis Martinez-Sobrido
  10. Wei Zheng

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Abstract

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  1. Version published to 10.1021/acsptsci.1c00182
  2. ScreenIT

    SciScore for 10.1101/2021.07.03.450938: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Reagents: Vero E6 cells (CRL-1586, Resource Research Identifier (RRID): CVCL0574) were purchased from American Tissue Type Collection.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Rabbit anti-NP SARS-CoV-2 antibody R001 (40143-R001, RRID Number: AB_2827974), R004 (40143-R004, RRID Number: AB_2827975), R019 (40143-R019, RRID Number: AB_2827973), and R040 (40143-R040, RRID Number: AB_2827976) was purchased from Sino Biological and mouse anti-NP MAb 1C7C7 was provided by Dr. Tomas Moran and purchased from Leinco (LT7000).
    anti-NP SARS-CoV-2
    detected: (Sino Biological Cat# 40143-R019, RRID:AB_2827973)
    suggested: None
    R040
    suggested: (Sino Biological Cat# 40143-R040, RRID:AB_2827976)
    anti-NP
    suggested: None
    IgG antibodies R001, R004, R019, R040 were raised against recombinant SARS-CoV nucleocapsid phosphoprotein (NP (Sino Biological, 0143-V08B) and expressed in HEK293 cells.
    R019 , R040 were raised against recombinant SARS-CoV nucleocapsid phosphoprotein ( NP
    suggested: None
    0143-V08B
    suggested: None
    Then antigen-antibody complexes were detected using appropriate anti species HRP-conjugates and West Femto substrate.
    anti species HRP-conjugates
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    IgG antibodies R001, R004, R019, R040 were raised against recombinant SARS-CoV nucleocapsid phosphoprotein (NP (Sino Biological, 0143-V08B) and expressed in HEK293 cells.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Preparation of Viral Lysates and Tissue Culture Supernatant (TCS) Production: Vero-E6 cells were plated in 12-well plates at 450 000 cells/well in 1.25 mL of growth media.
    Vero-E6
    suggested: None
    PCR fragments were digested with SacI and SmaI and cloned into SacI- and SmaI-digested pCAGGS plasmid containing a C-terminal HA epitope tag (pCAGGS NP-HA) 20 uL of 15,000 Vero E6 cells per well was seeded into half-area 96-well plates and incubated O/N at 37°C, 5% CO2.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    PCR fragments were digested with SacI and SmaI and cloned into SacI- and SmaI-digested pCAGGS plasmid containing a C-terminal HA epitope tag (pCAGGS NP-HA) 20 uL of 15,000 Vero E6 cells per well was seeded into half-area 96-well plates and incubated O/N at 37°C, 5% CO2.
    pCAGGS
    suggested: RRID:Addgene_18926)
    A plasmid encoding for SARS-CoV-2 NP (pCAGGS NP-HA) was transfected into the cells using Lipofectamine 2000 according to manufacturer’s recommendations (starting concentration 100 ng of plasmid and two-fold serial dilutions).
    pCAGGS NP-HA
    suggested: None
    Software and Algorithms
    SentencesResources
    The custom labeling of MAbs was performed by Columbia Biosciences.
    Columbia Biosciences
    suggested: (Columbia Biosciences Corporation, RRID:SCR_004347)
    Nucleotide sequences were translated to amino acid sequences using ExPASY online software 31 and the codon frame that contained the full NP was copied to ClustalO (1.2.4)32 for multiple sequence alignment.
    ExPASY
    suggested: None
    Graphpad Prism V9.1.0 was used to generate all graphs.
    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: 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.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.07.03.450938v1 on bioRxiv