Rapid quantitative screening assay for SARS-CoV-2 neutralizing antibodies using HiBiT-tagged virus-like particles

  1. Kei Miyakawa
  2. Sundararaj Stanleyraj Jeremiah
  3. Norihisa Ohtake
  4. Satoko Matsunaga
  5. Yutaro Yamaoka
  6. Mayuko Nishi
  7. Takeshi Morita
  8. Ryo Saji
  9. Mototsugu Nishii
  10. Hirokazu Kimura
  11. Hideki Hasegawa
  12. Ichiro Takeuchi
  13. Akihide Ryo

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Abstract

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  1. Version published to 10.1093/jmcb/mjaa047
  2. ScreenIT

    SciScore for 10.1101/2020.07.20.20158410: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics statement: This study was approved by Yokohama City University Certified Institutional Review Board (Reference No. B200200048, B160800009), and the protocols used in the study were approved by the ethics committee.
    Consent: Written informed consent was obtained from the patient or family/guardian.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were probed with anti-SARS-CoV-2 Spike S2 (clone 1A9, GeneTex #632604) or anti-HIV p24 (clone 183-H12-5C, NIH AIDS Reagent Program #3537) antibodies and horseradish peroxidase-conjugated secondary antibodies (Cytiva).
    anti-SARS-CoV-2 Spike S2
    suggested: (Thermo Fisher Scientific Cat# PA5-112048, RRID:AB_2866784)
    anti-HIV
    suggested: (Dr. Bruce Chesebro and Dr. Hardy Chen - National Institute of Allergy and Infectious Diseases Cat# 183-H12-5C, RRID:AB_2819250)
    horseradish peroxidase-conjugated secondary antibodies (Cytiva).
    suggested: None
    In some experiments, anti-spike neutralizing antibody (1:100, Sino Biological #40592-MM57) or Camostat mesilate (20 µM, Wako #039-17761) was added to the mixture instead of serum.
    anti-spike neutralizing antibody ( 1:100 , Sino Biological #40592-MM57 )
    suggested: None
    anti-spike neutralizing antibody
    suggested: (Sino Biological Cat# 40592-MM57, RRID:AB_2857935)
    Wells were then washed with PBS-T (PBS with 0.05% Tween-20) and added with HPR-conjugated goat anti-human IgM or IgG antibody, incubated for 1 hour at room temperature.
    HPR-conjugated goat anti-human IgM
    suggested: None
    IgG antibody, incubated for 1 hour at room
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and plasmids: HEK293 cells (ATCC #CRL-1573) and VeroE6/TMPRSS2 cells (JCRB #1819) were cultured in DMEM containing 10% FBS.
    HEK293
    suggested: None
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Neutralizing assay using hiVLP-SARS2 (hiVNT): VeroE6/TMPRSS2-LgBiT cells seeded in 96-well plates were washed and inoculated with hiVLP-SARS2 stocks (50 µl) containing diluted serum (1:20 for Figs. 2A and 3, 1:20 to 1:12500 dilution for Fig. 2B).
    VeroE6/TMPRSS2-LgBiT
    suggested: None
    Software and Algorithms
    SentencesResources
    Neutralizing titer: The neutralizing titer (NT50) was calculated based on the half-maximal inhibitory concentrations determined by Prism 8.3.1 software (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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 10.1101/2020.07.20.20158410v1 on medRxiv