Rapid characterization of spike variants via mammalian cell surface display
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SciScore for 10.1101/2021.03.30.437622: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Antibodies were purified as described below or purchased from the following manufacturers: Mouse anti-FLAG M2 (Sigma-Aldrich; F3165); Goat anti-Mouse IgG(H+L), Human ads-Alexa Fluor® 488 (SouthernBiotech; anti-FLAGsuggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)anti-Mouse IgG(H+Lsuggested: NoneAnti-FLAG signal was also used as an internal normalization control to correct for changes in transfection efficiency and spike expression when measuring antibody or ACE2 binding. ACE2suggested: NoneCells were then washed with PBS-BSA and stained with secondary antibodies (5 μM Alexa Fluor® 488 anti-mouse … SciScore for 10.1101/2021.03.30.437622: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Antibodies were purified as described below or purchased from the following manufacturers: Mouse anti-FLAG M2 (Sigma-Aldrich; F3165); Goat anti-Mouse IgG(H+L), Human ads-Alexa Fluor® 488 (SouthernBiotech; anti-FLAGsuggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)anti-Mouse IgG(H+Lsuggested: NoneAnti-FLAG signal was also used as an internal normalization control to correct for changes in transfection efficiency and spike expression when measuring antibody or ACE2 binding. ACE2suggested: NoneCells were then washed with PBS-BSA and stained with secondary antibodies (5 μM Alexa Fluor® 488 anti-mouse (SouthernBiotech; 1031-30) and 10 μM Alexa Fluor® 647 anti-human (SouthernBiotech; 2048-31) in PBS-BSA), shaking at 950 rpm for 1 hr at 4°C. anti-humansuggested: (SouthernBiotech Cat# 2048-31, RRID:AB_2795692)Membranes were blocked in LICOR Odyssey Blocking Buffer (Neta Scientific) incubated with the following primary antibodies overnight: mouse anti-FLAG M2 antibody (1:10,000; Sigma-Aldrich; F3165) for spike protein or RBD detection and mouse anti-beta tubulin antibody (1:5000; Thermo Fisher; MA5-16308) as a loading control. anti-beta tubulinsuggested: NoneThe membrane was washed three times with PBST (1X PBS, 0.1% Tween 20) for 10 min and then incubated with the following secondary antibody for 2 hrs: goat anti-mouse IRDYe® 680 conjugated antibody (1:10,000; abcam; ab216776). anti-mousesuggested: NoneThe assay went through the following steps: 1) baseline: 60 s with BLI buffer; 2) IgG immobilizing: 360 s with anti-FLAG IgG; 3) spikes loading: 360 s with diluted supernatants; 4) baseline: 300 s with BLI buffer; 5) association: 600 s with serial diluted analytes (antibodies or ACE2); 6) dissociation: 600 s with BLI buffer. anti-FLAG IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Expi293 cells were cultured in Expi293 Expression Medium (Sigma-Aldrich; A1435101) and maintained in a humidified atmosphere of 8% CO2 and 37 °C while shaking continuously at 125 rpm. Expi293suggested: RRID:CVCL_D615)The HIV particles pseudotyped with SARS-CoV-2 spike variants D614G or B.1.1.7 were generated in HEK293T cells, following previously published protocols68. HEK293Tsuggested: NoneThe mixture was then added to HEK-293T target cells, stably expressing human ACE2 in 96-well white plates with a clear bottom. HEK-293Tsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Cells were transiently co-transfected with plasmids for (1) HIV virion formation proteins (HDM-Hgpm2, pRC-CMV-Rev1b, and HDM-tat1b; (2) one of the envelope proteins (2019-nCoV Spike-D614G mutant, B.1.1.7 variant or VSV-G) and (3) the lentiviral backbone expressing luciferase reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W). Spike-D614Gsuggested: NoneSoftware and Algorithms Sentences Resources Data were analyzed using FlowJo v9. FlowJosuggested: (FlowJo, RRID:SCR_008520)Half-maximal inhibitory concentrations (IC50) were calculated using a 3-parameter logistic regression equation (GraphPad Prism v9.0). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)EDTA-free Protease Inhibitor Cocktail; Millipore Sigma). Cocktail; Millipore Sigmasuggested: NoneParticle stacks were then imported into cryoSPARC v3.1.0 for 2D classification, ab initio 3D reconstruction, heterogeneous 3D refinement, and homogeneous 3D refinement70. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)We then used the Biopython Bio. Biopythonsuggested: (Biopython, RRID:SCR_007173)We then used the python parasail package (https://github.com/jeffdaily/parasail-python) to perform a semi-global alignment of each sequence to the reference sequence71. pythonsuggested: (IPython, RRID:SCR_001658)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 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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