Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS‐CoV‐2 spike protein

SciScore for 10.1101/2020.08.24.20180661: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	Consent: Each participant provided written consent to participate in the study which was performed according to the EU guidelines and the Declaration of Helsinki.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	Contamination: HEK293T cells expressing ACE2 were generated by lentiviral transduction with vector CSIB and selection in blasticidin S 8 All cell lines were routinely tested for the absence of mycoplasma.

Table 2: Resources

Antibodies
Sentences	Resources
Transduced cells were selected by FACS sorting 48 h later using the anti-EGFR antibody.	anti-EGFR suggested: None
The …

SciScore for 10.1101/2020.08.24.20180661: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	Consent: Each participant provided written consent to participate in the study which was performed according to the EU guidelines and the Declaration of Helsinki.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	Contamination: HEK293T cells expressing ACE2 were generated by lentiviral transduction with vector CSIB and selection in blasticidin S 8 All cell lines were routinely tested for the absence of mycoplasma.

Table 2: Resources

Antibodies
Sentences	Resources
Transduced cells were selected by FACS sorting 48 h later using the anti-EGFR antibody.	anti-EGFR suggested: None
The cell pellet was finally resuspended in a 1:200 dilution of mouse anti-human IgG1 Fc-PE (Ref.: 9054-09, Southern Biotech) and a 1:300 dilution of the Brilliant Violet 421™ anti-human EGFR Antibody (Ref.: 352911, Biolegend) in PBS-BSA.	anti-human IgG1 suggested: (SouthernBiotech Cat# 9054-09, RRID:AB_2796628) anti-human EGFR suggested: (BioLegend Cat# 352911, RRID:AB_2562213)
For multiplexing, the following antibodies were used (all from Cytognos, S.L.): FITC-labelled anti-IgG1, PE-labeled anti-IgG2, APC-labelled anti-IgG3, APCC750 labelled anti-IgG4, PEcy7-labelled anti-IgM andPerCPcy5.5-labelled anti-lgA (for IgA1 and lgA2).	anti-IgG1 suggested: None anti-IgG2 suggested: None anti-IgG3 suggested: None anti-IgG4 suggested: None PEcy7-labelled anti-IgM andPerCPcy5.5-labelled anti-lgA (for IgA1 suggested: None anti-IgM suggested: None lgA2 suggested: None
Bound antibodies were detected by incubation with mouse anti-human IgG1 secondary antibody coupled to horse-radish-peroxidase (HRP) (Southern Biotech) diluted 1/6,000 in 1% BSA in PBS which was then detected using an ABTS substrate solution (Invitrogen).	horse-radish-peroxidase (HRP suggested: None
The nitrocellulose membrane was developed by ECL (Pierce) using PO-coupled mouse anti-human secondary antibodies (Southern Biotech).	anti-human secondary antibodies (Southern Biotech). suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Human embryonic kidney HEK293T cells (ATCC CRL-3216) and human hepatocellular carcinoma HepG2 cells (ATCC HB-8065) were maintained in DMEM supplemented with 10% FBS in a CO2 incubator.	HEK293T suggested: None
Lentiviral vector and Jurkat cell transduction: To express the full-length spike S protein of SARS-CoV2 we used the lentiviral vector based on the epHIV-7 plasmid that contains the truncated version of human EGFR (huEGFRt) that lacks the domains essential for ligand binding and tyrosine kinase activity described in Wang et al 9.	Jurkat suggested: TKG Cat# TKG 0209, RRID:CVCL_0065)
For transduction, lentiviral-transducing supernatants were produced from transfected packaging HEK-293T cells as described previously 10.	HEK-293T suggested: None
Polybrene (8 μg/mL) was added to the viral supernatants prior to transduction of ACE2+HEK293T cells.	ACE2+HEK293T suggested: None
A total of 3×105 MOLT-4 cells were plated on a P24 flat-bottom well 350 μL of DMEM and 350 μL of viral supernatant were added.	MOLT-4 suggested: None
Jurkat-S cells were collected by centrifugation and resuspended in PBS at the same concentration.	Jurkat-S suggested: None
HepG2 cells were labelled with Cell Trace Violet (CTV, Invitrogen) for 5 min at 37°C in PBS; Jurkat-S were labelled with CFDA-SE (CFSE, Invitrogen) under the same conditions.	HepG2 suggested: None
Software and Algorithms
Sentences	Resources
Samples were then washed and labeled cells were analyzed on a FACSCalibur or FACSCanto II flow cytometer (Becton-Dickinson) and the data were analyzed with FlowJo software (BD).	FACSCalibur suggested: None FlowJo suggested: (FlowJo, RRID:SCR_008520)

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).

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.

Results from TrialIdentifier: No clinical trial numbers were referenced.

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).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS‐CoV‐2 spike protein

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