A pseudotyped lentivirus-based assay to titer SARS-CoV-2 neutralizing antibodies in Mexico

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   ScreenIT

   Feb 4, 2022

   SciScore for 10.1101/2022.01.27.478128: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Samples were collected and used upon signed informed consent and anonymization.
   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 3 x105 cells were incubated on ice with mouse anti-human ACE2 monoclonal antibody conjugated to AF-647 (Santa Cruz Biotechnology, USA, cat.	anti-human ACE2 suggested: None
   A chimeric monoclonal antibody (Sino Biological, cat 40150-D001) was used to detected SARS-CoV-2-S as primary antibody, and a goat anti-human IgG conjugated to HRP (Bio-Rad, cat. 204005) was used as secondary antibody.	anti-human IgG sugges…

   SciScore for 10.1101/2022.01.27.478128: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Samples were collected and used upon signed informed consent and anonymization.
   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 3 x105 cells were incubated on ice with mouse anti-human ACE2 monoclonal antibody conjugated to AF-647 (Santa Cruz Biotechnology, USA, cat.	anti-human ACE2 suggested: None
   A chimeric monoclonal antibody (Sino Biological, cat 40150-D001) was used to detected SARS-CoV-2-S as primary antibody, and a goat anti-human IgG conjugated to HRP (Bio-Rad, cat. 204005) was used as secondary antibody.	anti-human IgG suggested: (SouthernBiotech Cat# 2040-05, RRID:AB_2795644)
   Experimental Models: Cell Lines
   Sentences	Resources
   , Vero (ATCC CCL-81), Vero E6 (ATCC CRL-1586) and Caco-2 (HTB-37) cells were obtained from ATCC and maintained in high-glucose DMEM (Caisson, cat.	Vero E6 suggested: None Caco-2 suggested: None
   , pCAG-HIVgp, and pCMV-SARS-CoV-2-S-RSV-REV were co-transfected at a 3:2:1 DNA ratio using the calcium phosphate method to confluent HEK-293T cells.	HEK-293T suggested: None
   Post-incubation, 25,000 Vero cells were added to each well and the plate was incubated for 24 h at 37 °C and 5% CO2.	Vero suggested: None
   Recombinant DNA
   Sentences	Resources
   Vector constructions: The pCAG-HIVgp and pCMV-VSV-G-RSV-REV plasmids were acquired through the Riken Institute BioResource Center53.	pCMV-VSV-G-RSV-REV suggested: None
   The pCMV-SARS-CoV-2-S-RSV-REV plasmid was produced by cloning the SARS-CoV-2 S sequence, obtained from pCMV14-3X-Flag-SARS-CoV-2 S which encodes codon optimized SARS-CoV-2 S protein lacking the last 19 amino acids at the C-terminal3, in place of the VSV-G gene, between the NheI and XbaI restriction sites.	pCMV-SARS-CoV-2-S-RSV-REV suggested: None pCMV14-3X-Flag-SARS-CoV-2 suggested: RRID:Addgene_145780)
   The pLenti-Nluc was produced by cloning the Nluc sequence amplified from pCCI-SP6-ZIKV-Nluc, between the XbaI and BamHI restriction sites within the pLentiCRISPR v2 backbone (cat. 52961, Addgene, Massachussets, USA.) and removing the Cas9 gene.	pLenti-Nluc suggested: None pCCI-SP6-ZIKV-Nluc suggested: None pLentiCRISPR v2 suggested: RRID:Addgene_169885)
   We produced the plasmid pCMV-REV by removing the envelope protein gene from pCMV-VSV-G-RSV-REV.	pCMV-REV suggested: None
   , pCAG-HIVgp, and pCMV-SARS-CoV-2-S-RSV-REV were co-transfected at a 3:2:1 DNA ratio using the calcium phosphate method to confluent HEK-293T cells.	pCAG-HIVgp suggested: None
   Western blot: The selective incorporation of SARS-CoV-2-S or VSV-G, and p24 proteins in VP was validated by western blot.	VSV-G suggested: RRID:Addgene_138479)
   Software and Algorithms
   Sentences	Resources
   At least 20,000 events were acquired per sample, and the data was analysed using FlowJo v.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: All statistical analyzes were performed using GraphPad Prism v.9 software and p-values < 0.05 were considered statistically significant.	GraphPad 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:

   • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
   • 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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   Version published to 10.1101/2022.01.27.478128 on bioRxiv

   Feb 2, 2022