Human Surfactant Protein D Binds Spike Protein and Acts as an Entry Inhibitor of SARS-CoV-2 Pseudotyped Viral Particles

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1. Version published to 10.3389/fimmu.2021.641360

   May 14, 2021
2. 

   ScreenIT

   Dec 19, 2020

   SciScore for 10.1101/2020.12.18.423418: (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
   In a parallel experiment, the wells were washed and then incubated with polyclonal rabbit anti-human SP-D primary antibody (0.5 μg/ml) (1:5000) for 2 h at room temperature.	anti-human SP-D suggested: None
   Next day, the wells were washed and then incubated with anti-sheep IgG-HRP antibodies or anti-His antibodies (Genetex, GTX628914, 0.5 μg/ml) (1:2000) for 2 h.	anti-sheep IgG-HRP suggested: None
   For the detection of RBD binding, the wells were further incubated …

   SciScore for 10.1101/2020.12.18.423418: (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
   In a parallel experiment, the wells were washed and then incubated with polyclonal rabbit anti-human SP-D primary antibody (0.5 μg/ml) (1:5000) for 2 h at room temperature.	anti-human SP-D suggested: None
   Next day, the wells were washed and then incubated with anti-sheep IgG-HRP antibodies or anti-His antibodies (Genetex, GTX628914, 0.5 μg/ml) (1:2000) for 2 h.	anti-sheep IgG-HRP suggested: None
   For the detection of RBD binding, the wells were further incubated with anti-mouse IgG antibody (Abcam, ab6728, 0.5 μg/ml) (1:2000) for 2 h.	anti-mouse IgG suggested: (Abcam Cat# ab6728, RRID:AB_955440)
   Following PBS washes, the cells were probed with goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody linked to Alexa Fluor 647 (Thermo Fisher Scientific) (0.6 μl/100 μl per tube) for 1 h at room temperature in dark.	anti-Rabbit IgG suggested: None
   For binding experiments using rfhSP-D, SARS-CoV-2 S1 protein containing a C-terminal His-tag (Acro; S1N-C52H3) (5 μg/ml) was tagged with anti-His antibody (Genetex; GT395) (1:100) at 4°C for 1h and followed by pre-incubation with a series of two-fold dilutions of rfhSP-D (10 μg/ml) or mock (cells only) at 4°C for 1h.	His-tag suggested: None anti-His suggested: None
   After washing, coverslips were blocked with 2% w/v BSA for 1h and incubated with ACE2 antibody [SN0754] (1:250) (GeneTex, GTX01160), followed by Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (1:500) (Thermo Fisher Scientific) for 1 h at room temperature in dark.	ACE2 suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589) GTX01160 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and treatments: Human embryonic kidney (HEK) 293T or HEK293T cells overexpressing ACE2 receptor (HEK293T-ACE2) were cultured in complete Gibco Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% v/v fetal bovine serum (FBS), 100 U/ml penicillin (Sigma-Aldrich) and 100 μg/ml streptomycin (Sigma-Aldrich), and left to grow at 37°C in the presence of 5% v/v CO2 for approximately 48 h before passaging.	HEK suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045) 293T suggested: None
   HEK293T cells were seeded one day before, and then transfected with the indicated plasmids using TransIT®-LT1 transfection reagent (Mirus).	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   HEK293T-ACE2 cells (HEK293T cells overexpressing ACE2 receptor) (0.5×105 cells) were preincubated with rfhSP-D (0, 5, 10 and 20 μg/ml) for 24 h and then washed twice with PBS.	HEK293T-ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Statistical Analysis: GraphPad Prism 6.0 software was used to generate all the graphs.	GraphPad 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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3. 

   Version published to 10.1101/2020.12.18.423418 on bioRxiv

   Dec 18, 2020