Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2

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   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.05.26.117549: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   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 inhibition studies, mouse α-SARS-CoV-2 spike antibody (GeneTex	α-SARS-CoV-2 suggested: None
   #GTX632604), affinity-purified polyclonal α-human-Ace-2 antibody (R&D Systems #AF933), and recombinant human Ace-2 (R&D Systems #933-ZN-010) were diluted in OptiMEM to the desired concentrations.	α-human-Ace-2 suggested: None
   After 30 min on ice, cells were rinsed with 1 mL FACS buffer and resuspended in 100 µL FACS buffer containing 5 µL donkey-α-goat IgG-PE …

   SciScore for 10.1101/2020.05.26.117549: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   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 inhibition studies, mouse α-SARS-CoV-2 spike antibody (GeneTex	α-SARS-CoV-2 suggested: None
   #GTX632604), affinity-purified polyclonal α-human-Ace-2 antibody (R&D Systems #AF933), and recombinant human Ace-2 (R&D Systems #933-ZN-010) were diluted in OptiMEM to the desired concentrations.	α-human-Ace-2 suggested: None
   After 30 min on ice, cells were rinsed with 1 mL FACS buffer and resuspended in 100 µL FACS buffer containing 5 µL donkey-α-goat IgG-PE secondary antibody.	IgG-PE suggested: None
   After 30 min on ice, samples were washed twice with 1 mL FACS buffer and resuspended in 100 µL of a 0.5% saponin solution containing 2 µL goat α-rabbit IgG-AF647 secondary antibody.	2 µL goat α-rabbit suggested: None
   Membranes were blocked in 5% non-fat dry milk in TBST, washed three times with TBST, and incubated for 1 h at room temperature with primary antibody mouse α-SARS-CoV-2 Spike (1:1000, GeneTex #GTX632604) or rabbit polyclonal α-VSV (1:5000, Imanis Life Sciences #REA005).	α-VSV suggested: None
   Membranes were washed three times with TBST and incubated for 1 h at room temperature with secondary antibody goat α-mouse IgG-HRP (Prometheus #20-304) or goat α-rabbit IgG-HRP (Prometheus #20-303) at 1:20,000.	α-mouse IgG-HRP suggested: None α-rabbit IgG-HRP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: African green monkey Vero cells (ATCC® CCL-81™), Vero-αHis (29), and baby hamster kidney BHK-21 cells (ATCC® CCL-10™) were maintained in high-glucose DMEM supplemented with 5% fetal bovine serum and 1X penicillin/streptomycin (complete media) at 37°C/5% CO2.	Vero suggested: None BHK-21 suggested: None
   Following selection, Vero-DSP cells were maintained in complete media supplemented with 5 µg/mL puromycin.	Vero-DSP suggested: None
   The virus was propagated in Vero-αHis cells by inoculating 80% confluent monolayers in 10-cm plates with 1 mL of virus.	Vero-αHis suggested: None
   Flow cytometry: Vero-DSP1-Puro cells were dislodged using Versene, counted, and transferred to microcentrifuge tubes (5×105 cells/tube was used for Ace-2 staining and 1.5×106 cells/tube was used for TMPRSS2 staining).	Vero-DSP1-Puro suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   To generate Vero-DSP1-Puro (Vero-DSP1) and Vero-DSP2-Puro (Vero-DSP2) were generated by transducing Vero cells with lentiviral vectors SFFV-DSP1-7-P2A-Puro or SFFV-DSP8-11-P2A-Puro encoding the Renilla Rluc8 luciferase-GFP split reporter DSP1 or DSP2 (GenBank EF446136.1) (20) under control of the spleen focus forming virus (SFFV) promoter and linked to the puromycin resistance gene via a P2A cleavage peptide.	Vero-DSP1-Puro (Vero-DSP1) suggested: None Vero-DSP2-Puro (Vero-DSP2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Statistical Analyses: Descriptive statistics, comparisons, and regression analyses were performed in Graph Pad Prism, v8.4.0 (San Diego, CA).	Graph Pad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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 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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2. 

   Version published to 10.1101/2020.05.26.117549v1 on bioRxiv

   May 27, 2020