Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies
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Abstract
The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~ 300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nM-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.
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SciScore for 10.1101/2020.10.13.338095: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal protocols and procedures were approved by the Georgia Tech Institutional Animal Care and Use Committee. Randomization not detected. Blinding General: Monoclonal antibody sequencing and functional tests were performed by blinded researchers. Power Analysis not detected. Sex as a biological variable Immunizations, Antigens, and B cell extraction: Six-week old female pathogen-free BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA) and housed in the Physiological Research Laboratory at the Georgia Institute of Technology. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Mice selected for high … SciScore for 10.1101/2020.10.13.338095: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal protocols and procedures were approved by the Georgia Tech Institutional Animal Care and Use Committee. Randomization not detected. Blinding General: Monoclonal antibody sequencing and functional tests were performed by blinded researchers. Power Analysis not detected. Sex as a biological variable Immunizations, Antigens, and B cell extraction: Six-week old female pathogen-free BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA) and housed in the Physiological Research Laboratory at the Georgia Institute of Technology. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Mice selected for high anti-ectodomain serum antibody levels were sacrificed by CO2 asphyxiation on day 30 followed by splenectomy for B cell extraction. anti-ectodomainsuggested: (M. Watanabe - Hokkaido University, Hokkaido, Japan Cat# anti ect BAI3, RRID:AB_2571550)Antibodies were isotyped using the Rapid ELISA Mouse mAbs Isotyping Kit (ThermoFisher Pierce) with anti-mouse heavy chain capture antibody (anti-IgG1, IgG2a, IgGb, IgG3, IgA and IgM) or antimouse light chain (kappa or lambda) and analyzed by SDS-PAGE and Superdex-200 size-exclusion chromatography. anti-mouse heavy chainsuggested: Noneanti-IgG1, IgG2asuggested: NoneIgGb, IgG3suggested: NoneIgMsuggested: Noneantimouse light chain (kappa or lambda)suggested: NoneAntibody sequencing: Hybridoma clones expressing anti-SARS-CoV-2 monoclonal antibodies were propagated in IMDM supplemented with antibiotics and 10% Low IgG FBS in static culture at 37°C with 5% CO2. 3-5 x 106 Cells from each clone were harvested by centrifugation at 2,000 x g for 10 min and washed once in 0.01 M PBS (pH 7.4). anti-SARS-CoV-2suggested: NoneSlides were counterstained in Mayer’s Hematoxylin (Polyscientific), blued in lithium carbonate (Polysciences, Inc.) and cover slipped with aqueous mounting medium (Polysciences, Inc.) Of the 13 antibodies tested, immunohistochemical staining of SARS-CoV-2 case control material was detected for two (1-3D2 and 1-3D7). aqueous mounting medium (Polysciences, Inc.suggested: NoneExperimental Models: Cell Lines Sentences Resources Human Expi293 cells (Thermo Scientific) were transfected with these constructs using FectoPro (PolyPlus Transfection) and the proteins purified from culture supernatants by immobilized metal affinity chromatography using HisTrap Excel nickel columns (Cytiva). Expi293suggested: RRID:CVCL_D615)Experimental Models: Organisms/Strains Sentences Resources Immunizations, Antigens, and B cell extraction: Six-week old female pathogen-free BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA) and housed in the Physiological Research Laboratory at the Georgia Institute of Technology. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Software and Algorithms Sentences Resources All data were processed and graphed using GraphPad Prism v.8. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Antibodies were isotyped using the Rapid ELISA Mouse mAbs Isotyping Kit (ThermoFisher Pierce) with anti-mouse heavy chain capture antibody (anti-IgG1, IgG2a, IgGb, IgG3, IgA and IgM) or antimouse light chain (kappa or lambda) and analyzed by SDS-PAGE and Superdex-200 size-exclusion chromatography. ThermoFisher Piercesuggested: NoneSlides were counterstained in Mayer’s Hematoxylin (Polyscientific), blued in lithium carbonate (Polysciences, Inc.) and cover slipped with aqueous mounting medium (Polysciences, Inc.) Of the 13 antibodies tested, immunohistochemical staining of SARS-CoV-2 case control material was detected for two (1-3D2 and 1-3D7). Polyscientificsuggested: NoneResults 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 7. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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