Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking

Article activity feed

1. 

   Version published to 10.1101/2021.06.02.446813v2 on bioRxiv

   Jun 30, 2021
2. 

   ScreenIT

   Jun 5, 2021

   SciScore for 10.1101/2021.06.02.446813: (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
   Antigen-specific B Cell Sorting: Cells were stained and mixed with DNA-barcoded antigens and other antibodies, and then sorted using fluorescence activated cell sorting (FACS).	Antigen-specific B suggested: None
   In brief, synthesized antibody-encoding DNA (~2 μg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800 μL of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra …

   SciScore for 10.1101/2021.06.02.446813: (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
   Antigen-specific B Cell Sorting: Cells were stained and mixed with DNA-barcoded antigens and other antibodies, and then sorted using fluorescence activated cell sorting (FACS).	Antigen-specific B suggested: None
   In brief, synthesized antibody-encoding DNA (~2 μg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800 μL of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra Biosciences).	antibody-encoding DNA suggested: None
   The secondary antibody, goat anti-human IgG conjugated to peroxidase, was added at 1:10,000 dilution in 1% milk in PBS-T to the plates, which were incubated for one hour at room temperature.	anti-human IgG suggested: None
   Plates were washed three times with PBS-T, and bound ACE2 was detected using HRP-conjugated anti-mouse Fc antibody and TMB substrate.	anti-mouse suggested: None
   RTCA Method for Initial Screening of Antibody Neutralizing Activity: To screen for neutralizing activity in the panel of recombinantly expressed antibodies, we used a high-throughput and quantitative RTCA assay and xCelligence RTCA HT Analyzer (ACEA Biosciences) that assesses kinetic changes in cell physiology, including virus-induced cytopathic effect (CPE).	CPE suggested: None
   An antibody was classified as fully neutralizing if it completely inhibited SARS-CoV-2-induced CPE at the highest tested concentration, while an antibody was classified as partially neutralizing if it delayed but did not fully prevent CPE at the highest tested concentration2, 22. Real-time Cell Analysis (RTCA) Neutralization Assay: To determine neutralizing activity of IgG, we used real-time cell analysis (RTCA) assay on an xCELLigence RTCA MP Analyzer (ACEA Biosciences Inc.	determine neutralizing activity of IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   For antibody expression, microscale transfection was performed (~1 mL per antibody) of CHO cell cultures using the Gibco ExpiCHO Expression System and a protocol for deep 96-well blocks (Thermo Fisher Scientific).	CHO suggested: None
   A suspension of 18,000 Vero-E6 cells in 50 μL of cell culture medium was seeded in each well, and the plate was placed on the analyzer.	Vero-E6 suggested: None
   Triplicate wells containing virus only (maximal CPE in the absence of antibody) and wells containing only Vero cells in medium (no-CPE wells) were included as controls.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Plaque Reduction Neutralization Test (PRNT): The virus neutralization with live authentic SARS-CoV-2 virus (USA-WA1) was performed in the BSL-3 facility of the Galveston National Laboratory using Vero E6 cells (ATCC CRL-1586) following the standard procedure.	Vero E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   ) sequencing core at an appropriate target concentration for 10X Genomics library preparation and subsequent sequencing.	Genomics suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)
   Flow cytometry data were analyzed using FlowJo.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   In brief, synthesized antibody-encoding DNA (~2 μg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800 μL of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra Biosciences).	Integra Biosciences suggested: None
   For antibodies, the inhibitory concentration at 50% (IC50) values were calculated in Prism software (GraphPad) by plotting the midway point between the upper and lower plateaus of the neutralization curve among dilutions.	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Cryogenic Electron Microscopy (Cryo-EM): Motion correction, CTF estimation, particle picking, and 2D classification were performed using cryoSPARC v3.2.026.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
   ANOVA analysis was performed for neutralization potency comparisons using GraphPad Prism version 8.0.0.	GraphPad Prism suggested: (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.
   • 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.

   ---

   Results from scite Reference Check: We found no unreliable references.

   ---

   Read the original source
3. 

   Version published to 10.1101/2021.06.02.446813v1 on bioRxiv

   Jun 2, 2021