Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Version published to 10.1021/acsomega.0c03512

Dec 21, 2020

SciScore for 10.1101/2020.06.14.150607: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	Consent: After obtaining informed consent, serum was obtained by venipuncture (BD Vacutainer, serum), centrifuged, aliquoted and stored at -80°C prior to use. IRB: Protocol approval was obtained by the Institutional Review Board (protocol IRB# 2016-6137) of the Albert Einstein College of Medicine.
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
COVID-19 convalescence sera samples (2, 3, 5, 6, 7) were previously validated to be negative with RT-PCR for SARS-CoV-2 and positive ELISA for antibodies against SARS-CoV-2.	SAR…

SciScore for 10.1101/2020.06.14.150607: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	Consent: After obtaining informed consent, serum was obtained by venipuncture (BD Vacutainer, serum), centrifuged, aliquoted and stored at -80°C prior to use. IRB: Protocol approval was obtained by the Institutional Review Board (protocol IRB# 2016-6137) of the Albert Einstein College of Medicine.
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
COVID-19 convalescence sera samples (2, 3, 5, 6, 7) were previously validated to be negative with RT-PCR for SARS-CoV-2 and positive ELISA for antibodies against SARS-CoV-2.	SARS-CoV-2 suggested: None
The arrays were rinsed with 5% milk-PBST briefly and incubated for 1 hour at room temperature with a fluorescently labeled secondary antibody (Alexa Fluor 647 labeled goat anti-human IgG (H+L) cross-adsorbed secondary antibody, Thermo Fisher Scientific, Cat # A21445) diluted 1:150 in 5% milk-PBST.	anti-human IgG suggested: (Molecular Probes Cat# A-21445, RRID:AB_2535862)
Cells were then incubated with a PE-labeled anti-6x His tag antibody (Abcam Cat # ab72467), to detect Spike protein binding.	anti-6x suggested: (Abcam Cat# ab72467, RRID:AB_1267596)
After 45 minutes, cells were pelleted and washed twice, and then incubated with an anti-HIS PE antibody, washed twice, and analyzed on a SONY Spectral Analyzer.	anti-HIS PE suggested: None
Expression of hACE2, mACE2, CD147, CD26, Siglec9, Siglec10, Ceacam1, and Ceacam5 were confirmed by antibody staining of transfected HEK293F cells.	CD147 suggested: None Siglec10 suggested: None Ceacam1 suggested: None Ceacam5 suggested: None
Antibodies used: hACE2 and mACE2 (RND Cat # AF933-SP), CD147 (Biolegend Clone HIM6), CD26 (Biolegend Clone BA5b), Siglec9 (Biolegend Clone K8), Siglec10 (Biolegend Clone FG6), Ceacam1 and Ceacam5 (Biolegend Clone ASL-32).	CD26 suggested: None Siglec9 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
HEK293F Transient Transfections and Culture: HEK 293 suspension cells were cultured in HEK Freestyle Media (Invitrogen) at 37 C in a humidified shaking platform incubator (Kuhner) with 5% CO2.	HEK 293 suggested: None
High-throughput Screening of Human Plasma Membrane Protein Library: The human plasma membrane protein library was transfected into HEK293F cells in 48-well format.	HEK293F suggested: None
Software and Algorithms
Sentences	Resources
Nickel eluates were concentrated by centrifugation in 100K concentrator (ThermoFisher Cat # 88533) by spinning at 1100 g for intervals of 8 minutes (if necessary) and further purified by gel filtration on a HiLoad™ 16/600 Superdex™ 200 column (GE) equilibrated with 50 mM TRIS, 100 mM ArgCl, 150 mM NaCl, 10% Glycerol, pH 8.0.	ThermoFisher Cat suggested: None
Protein concentration was determined using an extinction coefficient (1468500 M-1cm-1) estimated from amino acid sequence by Expasy online ProtParam tool[20]	ProtParam suggested: (ProtParam Tool, RRID:SCR_018087)
Protein concentration was determined using an extinction coefficient (33350 M-1cm-1), estimated from amino acid sequence by Expasy online ProtParam, and was further analyzed by SDS-PAGE.	Expasy online ProtParam suggested: None
The resulting supernatant was purified on an AKTA FPLC (GE Biosciences).	GE Biosciences suggested: None
Data was exported in Excel, and then analyzed and graphed by GraphPad Prism.	Excel suggested: None GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
The program Prism v8 was used to generate the AUC figures shown in the text and supplement.	Prism suggested: (PRISM, RRID:SCR_005375)
Absorption was measured at OD450 using a Synergy4 plate reader (Biotek), and data was analyzed using GraphPad Prism7.0 to calculate IC50 values.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

It is important to note the limitations of the current platform. In particular, we stress that at present this is a qualitative approach, as a number of variables can impact the ability to detect antibody reactivity, including patient titers for specific antigens and the relative affinities of an antigen-specific pool. Furthermore, it should be appreciated that the current platform is programmed to detect the capture of IgG antibodies; thus, the resultant signal (or lack thereof) for a particular antigen could be the consequence of prevalences between different isotypes (IgA, IgD, IgE, IgG, IgM), which are known to evolve during the course of infection and subsequent resolution. Although currently focused on three antigens, this platform can be readily expanded to study differential antibody responses to different SARS-CoV-2 antigens and subdomains of those antigens amongst individuals, which are actively being investigated by others [52-55]. We are currently working to include not only other antigens from SARS-CoV-2 (E protein, M protein, etc.), but also antigens from other coronaviruses that may be cross reactive with SARS-CoV-2 antibodies. The analysis of antibody reactivity to multiple SARS-CoV-2 and related antigens will provide broad insight into the humoral immune response to SARS-CoV-2. Collectively, we provide standards and metrics for high quality protein reagents that can yield comparable clinical, biological and structural data as we continue to combat the global ...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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

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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Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

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