A potent SARS-CoV-2 neutralizing antibody recognizing a conserved epitope with broad mutant variant and SARS-CoV activity

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   ScreenIT

   Feb 10, 2022

   SciScore for 10.1101/2022.02.06.479332: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Briefly, 96 well plates were coated with His-Tag capture antibody and incubated at 4°C overnight then blocked with 1% BSA in PBS for 1 hour at 37°C.	His-Tag suggested: None
   Serial dilutions of the selected antibodies were loaded in duplicates and incubated for 1 hour at room temperature, followed by the addition of biotinylated human ACE2.	ACE2 suggested: None
   Bead-based multiplex assay method for SARS-CoV-2 spike protein binding: A custom assay was designed using Magplex-Avidin …

   SciScore for 10.1101/2022.02.06.479332: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Briefly, 96 well plates were coated with His-Tag capture antibody and incubated at 4°C overnight then blocked with 1% BSA in PBS for 1 hour at 37°C.	His-Tag suggested: None
   Serial dilutions of the selected antibodies were loaded in duplicates and incubated for 1 hour at room temperature, followed by the addition of biotinylated human ACE2.	ACE2 suggested: None
   Bead-based multiplex assay method for SARS-CoV-2 spike protein binding: A custom assay was designed using Magplex-Avidin microspheres (Luminex), which were coated in anti-HIS antibody [Biotin] (GenScript A00613, mouse IgG1k clone 6G2A9) at 2.5 μg/mL, washed, and then each microsphere was coated with 2.5 μg/mL of either SARS-CoV-1 S1-HIS, MERS S1-HIS, and SARS-CoV-2 S1-HIS or SARS-CoV-2 RBD-HIS variant proteins (Sino Biological) to create bead stocks.	anti-HIS suggested: (GenScript Cat# A00613, RRID:AB_915572) mouse IgG1k suggested: (GenScript Cat# A00186, RRID:AB_914704) SARS-CoV-2 suggested: None S1-HIS suggested: None
   Antibodies were incubated with microspheres for 30 minutes at RT with shaking at 300 rpm, washed 3x with PBS, then incubated with Goat anti-Human IgG Fc Secondary Antibody PE, eBioscience (minimal cross-reactivity to bovine/horse/mouse serum proteins)(Thermo Fisher Scientific 12-4998-82) diluted 1:200 in PBS/1% BSAfor 30 minutes.).	anti-Human IgG suggested: (Thermo Fisher Scientific Cat# 12-4998-82, RRID:AB_465926)
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, the recommended amount of particles/well was incubated in DMEM + 10% FBS with varying amounts of serially-diluted antibody for 1 hour at 37°C, and then 20,000 ACE2-HEK293T cells (Integral Molecular, see Extended Table 5) were added.	ACE2-HEK293T suggested: None
   The mutant library was arrayed in 384-well microplates and transiently transfected into HEK-293T cells.	HEK-293T suggested: None
   Software and Algorithms
   Sentences	Resources
   Results were analyzed in GraphPad PRISM Version 9.3.0.	GraphPad PRISM suggested: (GraphPad Prism, RRID:SCR_002798)
   Structural analysis: The figures were rendered using PyMOL Version 2.4.1 (The PyMOL Molecular GraphicsSystem; http://www.pymol.Org).	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   Multiple alignment of related lineage B betacoronavirus spike protein sequences: Multiple alignment was made using Clustal Omega (Madeira, 2019) and image rendered using Geneious software (Kearse, 2012).	Clustal Omega suggested: (Clustal Omega, RRID:SCR_001591) Geneious suggested: (Geneious, RRID:SCR_010519)

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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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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

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   Version published to 10.1101/2022.02.06.479332 on bioRxiv

   Feb 7, 2022