A novel high-throughput single B-cell cloning platform for isolation and characterization of high-affinity and potent SARS-CoV-2 neutralizing antibodies
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SciScore for 10.1101/2022.03.20.485024: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. 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 Cells were successively stained for 30 minutes on ice with the cocktail of fluorescently conjugated antibodies in 200 μl staining buffer (1X PBS + 2% FBS): 2 μg/ml biotinylated-RBD-Streptavidin-PE and FITC-anti-human IgG antibody (Biolegend, clone: M1310G05) and DAPI. FITC-anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293T cells were seeded in a T-75 flask at a density of 4.5×106 cells/flask/15ml media. HEK293Tsuggested: NoneFor pseudovirus-based … SciScore for 10.1101/2022.03.20.485024: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. 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 Cells were successively stained for 30 minutes on ice with the cocktail of fluorescently conjugated antibodies in 200 μl staining buffer (1X PBS + 2% FBS): 2 μg/ml biotinylated-RBD-Streptavidin-PE and FITC-anti-human IgG antibody (Biolegend, clone: M1310G05) and DAPI. FITC-anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293T cells were seeded in a T-75 flask at a density of 4.5×106 cells/flask/15ml media. HEK293Tsuggested: NoneFor pseudovirus-based neutralization assay, HEK293 cells stably expressing ACE-2 receptor (HEK293-ACE2) were plated into a tissue culture treated opaque white 96-well microplate in complete medium at a density of 5000 cells/well. HEK293suggested: NoneAntibody and pseudovirus suspension was then added to the HEK293-ACE2 cells and 10μl of 50μg/ml (10X) of polybrene to the cells (5μg/ml final concentration). HEK293-ACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources The next day plasmid DNA was diluted in 500μl OptiMEM at a ratio of Transfer vector (CMV-GFP-T2A-Luciferase, BLIV101PA-1, System-bio) CMV-GFP-T2A-Luciferasesuggested: NoneRecombinant DNA Sentences Resources Samples showing the correct size band for heavy and light chain amplification in the respective wells were subsequently cloned into pAb20-hCHIgG1 (Synbio; for heavy chain) and pAb20-hCK (Synbio; for light chains) through in-fusion cloning (Takara). pAb20-hCHIgG1suggested: None24 hours later, pAb20-hCHIgG1 and pAb20-hCK plasmids separately expressing heavy and light chain of antibodies were transiently cotransfected into Freestyle-293F cells (R79007; Thermo Fisher) using purefection reagent (LV750A-1; System Bio) followed by manufacturer protocol. pAb20-hCKsuggested: Noneviral packaging (psPAX2): viral envelope (pMD2G) or Sars-CoV-2 envelope (pMT1-SARS-CoV-2-S, custom gene synthesis; Synbio) at 4:2:1 ratio (6:3:1.5ug, respectively). pMD2Gsuggested: NonepMT1-SARS-CoV-2-Ssuggested: NoneSoftware and Algorithms Sentences Resources Lastly, the half-maximal inhibitory concentrations (IC50) were measured using the four-parameter logistic regression in GraphPad Prism 8.0. 2.7 Statistical analysis: All grouped data are expressed as the mean ± standard deviation (SD) of a demonstrative experiment executed at least in triplicate, and almost similar data were obtained in at least three independent experiments. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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