IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers

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1. Version published to 10.1128/msphere.00170-21

   Jun 30, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.16.21251653: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Samples were obtained with informed consent and the protocol was conducted under ICH-GCP and all applicable sections of the Code of Federal Regulations.
   Randomization	Blinded Sample Testing: Sera and plasma samples were randomized by independent operators prior to being given to analysts for testing.
   Blinding	For specificity and sensitivity studies, each blinded sample was tested by four different analysts, on at least three different days, in a total of five separate assay runs, using two different virus lots.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resour…

   SciScore for 10.1101/2021.02.16.21251653: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Samples were obtained with informed consent and the protocol was conducted under ICH-GCP and all applicable sections of the Code of Federal Regulations.
   Randomization	Blinded Sample Testing: Sera and plasma samples were randomized by independent operators prior to being given to analysts for testing.
   Blinding	For specificity and sensitivity studies, each blinded sample was tested by four different analysts, on at least three different days, in a total of five separate assay runs, using two different virus lots.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   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 mouse monoclonal α-VSV-G clone 8G5F11 (1:10,000	α-SARS-CoV-2 suggested: None α-VSV-G 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) at 1:20,000.	α-mouse IgG-HRP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: African green monkey Vero cells (ATCC® CCL-81™), Vero-αHis (47), 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
   Vero-ACE2-Puro/TMPRSS2-Puro (Vero-ACE2/TMPRSS2) cells were generated by transducing Vero-ACE2-Puro cells with lentiviral vector SFFV-TMPRSS2-Puro encoding human TMPRSS2 cDNA (GenBank: BC051839) under control of the SFFV promoter and linked to the puromycin resistance gene via a P2A cleavage peptide.	Vero-ACE2-Puro suggested: None
   Following selection, Vero-ACE2 cells were maintained in complete media supplemented with 5 µg/mL puromycin.	Vero-ACE2 suggested: None
   Aliquots were used to determine viral titers by plaque assay on Vero-αHis cells.	Vero-αHis suggested: None
   Cell lysates from HEK-293T cells stably expressing SARS-CoV-2 spike protein were also prepared as controls.	HEK-293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Vero-ACE2-Puro (Vero-ACE2) cells were generated by transducing Vero cells with lentiviral vector LV-SFFV-ACE2-Puro, encoding the human ACE2 cDNA (GenBank BC039902) under control of the spleen focus forming virus (SFFV) promoter and linked to the puromycin resistance gene via a P2A cleavage peptide.	Vero-ACE2-Puro suggested: None
   Vero-ACE2-Puro/TMPRSS2-Puro (Vero-ACE2/TMPRSS2) cells were generated by transducing Vero-ACE2-Puro cells with lentiviral vector SFFV-TMPRSS2-Puro encoding human TMPRSS2 cDNA (GenBank: BC051839) under control of the SFFV promoter and linked to the puromycin resistance gene via a P2A cleavage peptide.	Vero-ACE2-Puro/TMPRSS2-Puro (Vero-ACE2/TMPRSS2) suggested: None
   Software and Algorithms
   Sentences	Resources
   Statistical Analyses: Descriptive statistics, comparisons, and regression analyses were performed in Graph Pad Prism, v9.0.0 (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 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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3. 

   Version published to 10.1101/2021.02.16.21251653v1 on medRxiv

   Feb 19, 2021