Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Version published to 10.1038/s41591-020-0998-x

Jul 10, 2020

SciScore for 10.1101/2020.05.12.091462: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IRB: The studies were approved by the Institutional Review Board of Vanderbilt University Medical Center, and subsite studies were approved by the Institutional Review Board of the University of Washington or the Research Ethics Board of the University of Toronto. Consent: Samples were obtained after written informed consent.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Subject 1 (35-year-old male) was the earliest reported case of SARS-CoV-2 infection in the U.S. who presented with disease in Seattle, WA on January 19, 20201, a blood sample was obtained for study on February 19, 2020.
Cell Line Authentication	Contamination: Mycoplasma testing of Expi293F and ExpiCHO cultures was performed on a monthly basis using a PCR-based mycoplasma detection kit (ATCC, 30-1012K).

Table 2: Resources

Antibodies
Sentences	Resources
Briefly, B cells were purified magnetically (STEMCELL Technologies) and stained with anti-CD19-APC (clone HIB19, 982406), -IgD-FITC (clone LA6-2, 348206), and -IgM-FITC (clone MNM-88, 314506) phenotyping antibodies (BioLegend) and biotinylated antigen.	anti-CD19-APC suggested: (Sigma-Aldrich Cat# SAB4700110, RRID:AB_10898737) -IgM-FITC suggested: (BioLegend Cat# 305210, RRID:AB_314506)
Assays consisted of mixing beads conjugated with the RBD-mFc or S2Pecto proteins with fluorescently-labeled anti-human secondary antibodies (AF568, Thermo Fisher Scientific) and importing this assay mixture into OptoSelect 11k chips.	anti-human secondary antibodies ( AF568 suggested: None
Antigen-specific antibodies bound the antigen-conjugated beads, which then sequestered the fluorescent secondary antibody.	Antigen-specific suggested: None
Sequencing and cloning of single antigen-specific B cells: After export from the Beacon, antibody heavy and light chain sequences for B cells secreting antibodies with RBD-mFc-or S2Pecto-binding antibodies were amplified and recovered using components of the Opto™ Plasma B Discovery cDNA Synthesis Kit (Berkeley Lights)	S2Pecto-binding suggested: None
Any antibody variant that was designated as an IgM isotype (based on the sequence and assignment using the 10x Genomics Cell Ranger V(D)J software [version 2.1.1]) was removed from consideration (while IgG and IgA isotype antibodies were retained).	IgA isotype antibodies were retained) . suggested: None
The bound antibodies were detected using goat anti-human IgG conjugated with HRP (horseradish peroxidase) (Southern Biotech) and TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Thermo Fisher Scientific).	anti-human IgG suggested: None
The plates were incubated sequentially with 1 µg/mL of rCR3022 anti-S antibody15 and horseradish-peroxidase (	anti-S suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell culture: Vero E6 (CRL-1586, American Type Culture Collection (American Type Culture Collection, ATCC), Vero CCL81 (ATCC), HEK293 (ATCC), and HEK293T (ATCC) were maintained at 37°C in 5% CO2 in Dulbecco’s minimal essential medium (DMEM) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/mL of penicillin–streptomycin.	HEK293 suggested: None HEK293T suggested: None
Virus was passaged in Vero CCL81 cells and titrated by plaque assay on Vero E6 cells.	Vero CCL81 suggested: None Vero E6 suggested: RRID:CVCL_XD71)
After recovered sequences were cloned into pMCis_G1 expression constructs, recombinant antibodies were expressed in Chinese hamster ovary (CHO) cells and purified by affinity chromatography as detailed below.	Chinese hamster ovary ( CHO ) suggested: None
For “midi-scale” mAbs expression, we performed transfection (∼15 mL per antibody) of CHO cell cultures using the Gibco™ ExpiCHO™ Expression System and protocol for 50 mL mini bioreactor tubes (Corning) as described by the vendor.	CHO suggested: None
Wells containing virus only (in the absence of mAb) and wells containing only Vero cells in medium were included as controls.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
Software and Algorithms
Sentences	Resources
Image processing was performed using the cryoSPARC software package6.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon® Ultra-4 50KDa Centrifugal Filter Units (Millipore Sigma) and stored at 4°C until use.	Amicon® suggested: None
Half-maximal effective concentration (EC50) values for binding were determined using Prism v8.0 software (GraphPad) after log transformation of mAb concentration using sigmoidal dose-response nonlinear regression analysis.	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Twenty (20) μL of cell culture medium (DMEM supplemented with 2% FBS) was added to each well of a 96-well E-plate using a ViaFlo384 liquid handler (Integra Biosciences) to obtain background reading.	Integra Biosciences suggested: None
The distribution of CDR3 amino acid lengths were then plotted as histograms and fitted using kernel density estimation for the curves using python seaborn library.	python suggested: (IPython, RRID:SCR_001658)
Data were analyzed using ForeCyt software version 6.2 (IntelliCyt Corp).	ForeCyt suggested: None

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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Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

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