A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

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1. Version published to 10.1038/s41467-021-25777-z

   Sep 17, 2021
2. 

   ScreenIT

   Nov 2, 2020

   SciScore for 10.1101/2020.10.29.361287: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	CDR2 was randomized in stage one, PCR templates at this stage were equal molar mixtures of plasmids carrying DNA encoding frames, including three frame1 versions, one frame2, three frame3 versions and one frame4.
   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
   ) with anti-Flag antibody (Sigma-Aldrich, F1804), then incubating antibody-coated beads with cell lysate or cell media containing 3xFlag tagged target proteins at 4°C for 2 hours.	anti-Flag suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044) F1804 suggested: None

   SciScore for 10.1101/2020.10.29.361287: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	CDR2 was randomized in stage one, PCR templates at this stage were equal molar mixtures of plasmids carrying DNA encoding frames, including three frame1 versions, one frame2, three frame3 versions and one frame4.
   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
   ) with anti-Flag antibody (Sigma-Aldrich, F1804), then incubating antibody-coated beads with cell lysate or cell media containing 3xFlag tagged target proteins at 4°C for 2 hours.	anti-Flag suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044) F1804 suggested: None
   For anti-Myc selection, magnetic beads were coated by anti-Myc antibody (ThermoFisher Scientific, 13-2500) only.	anti-Myc suggested: None
   Plates were washed three times with wash buffer, HRP conjugated anti-His tag secondary antibody (BioLegend, 652503) diluted 1:2000 in blocking buffer was then added to the plates and incubated at RT for 1 hour.	anti-His tag secondary antibody ( BioLegend , 652503 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T ACE2 were a kind gift of Michael Farzan.	HEK293T ACE2 suggested: RRID:CVCL_YZ65)
   Plates were washed once with PBST (PBS, ThermoFisher Scientific, with 0.02% TritonX-100), a 1:1 mixture of HEK293T cell culture media containing secreted RBD-3xFlag and blocking buffer (PBST with 1% nonfat dry milk) was added to the plates and incubated at RT for 1 hour.	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Medium was then removed from HEK293T ACE2/TMPRSS2 cells and replaced with 150 μl of the VHH + pseudotyped lentivirus solution.	HEK293T ACE2/TMPRSS2 suggested: None
   Recombinant DNA
   Sentences	Resources
   psPAX2 and pCMV-VSV-G were previously described 20. pTRIP-SFFV-EGFP-NLS was previously described 21 (a gift from Nicolas Manel; Addgene plasmid # 86677; http://n2t.net/addgene:86677; RRID:Addgene_86677). cDNA for human TMPRSS2 and Hygromycin resistance gene was obtained by synthesis (IDT).	detected: RRID:Addgene_86677)
   Software and Algorithms
   Sentences	Resources
   CDR-directed clustering analysis: Computational analysis for CDR-directed clustering was performed using custom python scripts.	python suggested: (IPython, RRID:SCR_001658)
   Percentage GFP was quantified on a Cytoflex LX (Beckman Coulter) and data were analyzed with FlowJo.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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   Results from OddPub: Thank you for sharing your code.

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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 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).

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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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3. 

   Version published to 10.1101/2020.10.29.361287v1 on bioRxiv

   Oct 30, 2020