Potent mouse monoclonal antibodies that block SARS-CoV-2 infection

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1. 

   ScreenIT

   Oct 4, 2020

   SciScore for 10.1101/2020.10.01.323220: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Sample collection is approved by Keio University Bioethics Committee with the number 20200063.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For antibody production, monoclonal hybridomas were cultured in Hybridoma Serum-Free Medium (FUJIFILM Wako) supplemented with IL-6.	IL-6 suggested: None
   After three times washing in PBS-T (0. 1% Tween-20), the membrane was incubated in 1:5000 dilution of the peroxidase-conjugated sheep anti-mouse IgG secondary antibody (MP Biomedicals) for 30 min at room temperature.	anti-mou…

   SciScore for 10.1101/2020.10.01.323220: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Sample collection is approved by Keio University Bioethics Committee with the number 20200063.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   For antibody production, monoclonal hybridomas were cultured in Hybridoma Serum-Free Medium (FUJIFILM Wako) supplemented with IL-6.	IL-6 suggested: None
   After three times washing in PBS-T (0. 1% Tween-20), the membrane was incubated in 1:5000 dilution of the peroxidase-conjugated sheep anti-mouse IgG secondary antibody (MP Biomedicals) for 30 min at room temperature.	anti-mouse IgG suggested: None
   Monoclonal antibodies starting from 100 µg/mL were four-folds serial diluted with blocking buffer to 12 gradients and incubated with plates for 1 hour at room temperature, followed by incubation with horseradish peroxidase (HRP) conjugated sheep anti-mouse secondary antibody (MP Biomedicals) 1:5000 diluted in blocking buffer for 30 min at room temperature.	anti-mouse suggested: None
   ACE2-binding inhibition assay: For spike pull-down assay, SΔTM glycoprotein was incubated with 1 µg anti-spike antibody in 50 µl binding buffer (PBS supplemented with 0.1% NP-40) at room temperature for 1 hour, then 3 µg of ACE2-SBP recombinant protein was applied the reaction for 1 hour.	anti-spike suggested: None
   After washing, beads were incubated with diluted antibodies for 20 min at 4°C, washed, incubated with 4 µg/mL of ACE2-FLAG for 20 min at 4°C, washed, and incubated with an anti-DYKDDDDK antibody conjugated with APC fluorophore (MBL) for 20 min at 4°C.	anti-DYKDDDDK suggested: None
   Mouse anti-FLAG M2 antibody (Sigma) was also used as a control.	anti-FLAG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell cultures: The mouse myeloma cell line SP2/0-Ag14 (RCB0209) was provided by the Riken Bioresouces Center (Tsukuba, Japan).	SP2/0-Ag14 suggested: RCB Cat# RCB0209, RRID:CVCL_2199)
   Splenocytes (1×108) were immediately mixed with 5×107 SP2/0 myeloma cells and fused using an electro cell fusion generator ECFG21 (NepaGene) according to the manufacturer’s instructions.	SP2/0 suggested: None
   Lysates of 293T cells transfected with plasmids encoding full-length spike glycoproteins was also separated by SDS-PAGE for WB.	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Immunoprecipitation of SΔTM was examined by SDS-PAGE followed by western blotting using antibody R52. Immunofluorescence: Before performing immunofluorescence, HeLa cells seeded on cover glasses were transfected with plasmids encoding full length SARS-CoV-2 spike protein for 2 days using Lipofectamine 2000 (Thermo Fisher).	HeLa suggested: None
   Virus neutralization assay: SARS-CoV-2 virus (obtained from the National Institute of Infectious Diseases) was prepared from culture fluids harvested from infected VeroE6/TMPRSS2 cells (JCRB Cell Bank, JCRB1819) (Matsuyama et al., 2020).	VeroE6/TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Production of monoclonal antibodies: BALB/c mice were immunized twice in 3-week intervals, with the second immunization serving as a booster.	BALB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   Signal was quantified by measuring absorbance at 450 nanometer using iMark Microplate Absorbance Reader (Bio-Rad Laboratories).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.

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2. 

   Version published to 10.1101/2020.10.01.323220 on bioRxiv

   Oct 2, 2020