Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants

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   ScreenIT

   Sep 13, 2021

   SciScore for 10.1101/2021.09.10.459744: (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
   ELISA experiments: 96-well ELISA plates were coated with 100μL of anti-ACE2 coating antibody (2μg/ml diluted in PBS pH 7.4) over night at room temperature.	anti-ACE2 suggested: None
   Following incubation for 1 hour at room temperature, plates were washed five times with washing buffer after which 100μL of anti-Histidine detection antibody (diluted 1/500 in blocking buffer) was added for another hour.	anti-Histidine suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Surface …

   SciScore for 10.1101/2021.09.10.459744: (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
   ELISA experiments: 96-well ELISA plates were coated with 100μL of anti-ACE2 coating antibody (2μg/ml diluted in PBS pH 7.4) over night at room temperature.	anti-ACE2 suggested: None
   Following incubation for 1 hour at room temperature, plates were washed five times with washing buffer after which 100μL of anti-Histidine detection antibody (diluted 1/500 in blocking buffer) was added for another hour.	anti-Histidine suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Surface Plasmon Resonance Analysis: Recombinant SARS-CoV-2 spike proteins (His-tagged) were purchased from Acro Biosystems Inc. (Newark, USA) or produced inhouse by overexpression of respective constructs in HEK293T cells followed by purification via nickel NTA chromatography and size exclusion chromatography.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Vero E6 cells were cultured in DMEM medium (Gibco, Gaithersburg, MD, USA or Thermofisher) containing 10% fetal bovine serum (FBS).	Vero E6 suggested: None
   Calu-3 cells (HTB-55; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in DMEM F12 Medium (ATCC) with 20% FBS.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Mutations in isolates used at the Karolinska are as follows (with Spike mutations indicated in bold): Preparation of recombinant human ACE2: Clinical grade recombinant human ACE2 (amino acids 18-740) was produced by the contract manufacturer Polymun Scientific (Klosterneuburg, Austria) from CHO cells according to GMP guidelines under serum free conditions and formulated as a physiologic aqueous solution, as described previously (Haschke et al	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Using BiaEvaluation 4.1 software, kinetic analysis was carried out by applying a Langmuir 1:1 (apparent affinity) and a bivalent analyte binding algorithm, which separates first step binding (A + B = AB; affinity) from the second step binding (AB + B = AB2; avidity)	AB suggested: RRID:BDSC_203)
   Software and Algorithms
   Sentences	Resources
   Using BiaEvaluation 4.1 software, kinetic analysis was carried out by applying a Langmuir 1:1 (apparent affinity) and a bivalent analyte binding algorithm, which separates first step binding (A + B = AB; affinity) from the second step binding (AB + B = AB2; avidity)	BiaEvaluation suggested: (BIAevaluation Software, RRID:SCR_015936)
   Half-maximal inhibitory concentration (IC50) and inhibitory concentration 90 (IC90) were calculated using GraphPad Prism Software (La Jolla, CA)	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Visualizations of RBDs and full-length Spike protein: Visualizations were rendered with pymol software (the PyMOL Molecular Graphics System, Version 2.4 Schrödinger, LLC), based on a model of the fully glycosylated Spike-ACE2 complex described in Capraz et al., (submitted for publication) and https://covid.molssi.org//models/#spike-protein-in-complex-with-human-ace2-ace2-spike-binding. Primers and Antibodies: The following tables lists the primers and Antibodies used in this study:	PyMOL suggested: (PyMOL, RRID:SCR_000305)

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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: We detected the following sentences addressing limitations in the study:

   Limitations of this study: Our study used two different cell types and should be expanded using additional human cell types and also organoids. Moreover, to indeed make a claim on universality, additional variants should (and can) be tested using affinity/avidity measurements and neutralization assays.

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04335136	Completed	Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a…

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

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2. 

   Version published to 10.1101/2021.09.10.459744v1 on bioRxiv

   Sep 10, 2021