Rapid generation of potent antibodies by autonomous hypermutation in yeast
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SciScore for 10.1101/2020.11.11.378778: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Between 2 x 109 and 1 x 1010 induced yeast were pelleted and resuspended in 2.5-5 mL of AT1R staining buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 3 mM CaCl2, 0.1% MNG, 0.01% CHS, 0.2% maltose, 20 µM angiotensin II) with 1 µM anti-HA antibody that was fluorescently labeled, alternately, with AlexaFluor (AF) 647 or FITC. angiotensin II) with 1suggested: Noneanti-HAsuggested: (Immune Biosolutions Cat# Y00001-647, RRID:AB_2622007)Following this, yeast … SciScore for 10.1101/2020.11.11.378778: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Between 2 x 109 and 1 x 1010 induced yeast were pelleted and resuspended in 2.5-5 mL of AT1R staining buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 3 mM CaCl2, 0.1% MNG, 0.01% CHS, 0.2% maltose, 20 µM angiotensin II) with 1 µM anti-HA antibody that was fluorescently labeled, alternately, with AlexaFluor (AF) 647 or FITC. angiotensin II) with 1suggested: Noneanti-HAsuggested: (Immune Biosolutions Cat# Y00001-647, RRID:AB_2622007)Following this, yeast cells were again pelleted and stained with fluorescently labeled anti-FLAG antibody and fluorescently labeled anti-HA antibody for 20 min at 4°C, alternating between FITC, 647, and 488 labeled antibody for each AHEAD cycle to avoid selection for dye binding. anti-FLAGsuggested: (Immune Biosolutions Cat# Y00004-647, RRID:AB_2622019)For the first round of FACS, 1 x 108 induced cells were stained with 1 µM of directly AF488-labeled SARS-CoV-2 RBD and 0.5 µM anti-HA AF647 (Cell Signaling Technology) antibody, to visualize expression, for 1 hr at 4°C. anti-HA AF647 (Cell Signaling Technology)suggested: NoneCells were then washed and incubated with 0.5 µM goat anti-mouse AF488-labeled antibody (ThermoFisher) for 15 minutes. anti-mouse AF488-labeledsuggested: NoneExperimental Models: Cell Lines Sentences Resources To delete MET17, F102-2 cells were transformed with a linear DNA fragment encoding a G418 resistance cassette flanked on both sides by 45bp sequences homologous to the surrounding regions of MET17 (SG ID S000004294). F102-2suggested: NoneFor each nanobody-Fc fusion, 100 mL of Expi293 cells were transfected with 90-150 µg of plasmid. Expi293suggested: RRID:CVCL_D615)Pseudotypes were packaged by transfecting HEK293T cells (ATCC CRL-11268) using lipofectamine 3000 (Invitrogen) with SARS-CoV-2 S in pCAGGS or VSV G in pCAGGS (as previously described (34)), in addition to a packaging vector containing HIV Gag, Pol, Rev, and Tat (psPAX2, provided by Didier Trono, Addgene # 12260), and a pLenti transfer vector containing GFP (pLenti-EF1a-Backbone, a gift from Feng Zhang (35), Addgene plasmid #27963). HEK293Tsuggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)Software and Algorithms Sentences Resources Affinity maturation of Nb.b201 and Lag42 using the second-generation AHEAD 2.0 system: HSA (Sigma) was directly labeled with AF647. 6XHis tagged GFP was expressed in E. coli and purified using Ni-NTA agarose (ThermoFisher). AHEADsuggested: (AHEAD, RRID:SCR_008890)Nanobody-Fc fusion supernatants were passed over a column with 4 mL protein G resin (ThermoFisher), which was then washed with 40 mL of HBS, eluted with 100 mM citrate (pH 3) and then neutralized to pH 7 with concentrated HEPES (pH 8). ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Average PE signal at each antigen concentration was determined and used to fit a one-site model in GraphPad Prism in order to determine the EC50. 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 26, 28 and 31. 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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SciScore for 10.1101/2020.11.11.378778: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Between 2 x 109 and 1 x 1010 induced yeast were pelleted and resuspended in 2.5-5 mL of AT1R staining buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 3 mM CaCl2, 0.1% MNG, 0.01% CHS, 0.2% maltose, 20 µM angiotensin II) with 1 µM anti-HA antibody that was fluorescently labeled, alternately, with AlexaFluor (AF) 647 or FITC. angiotensin II ) with 1suggested: None…SciScore for 10.1101/2020.11.11.378778: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Between 2 x 109 and 1 x 1010 induced yeast were pelleted and resuspended in 2.5-5 mL of AT1R staining buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 3 mM CaCl2, 0.1% MNG, 0.01% CHS, 0.2% maltose, 20 µM angiotensin II) with 1 µM anti-HA antibody that was fluorescently labeled, alternately, with AlexaFluor (AF) 647 or FITC. angiotensin II ) with 1suggested: NoneFollowing this, yeast cells were again pelleted and stained with fluorescently labeled anti-FLAG antibody and fluorescently labeled anti-HA antibody for 20 min at 4°C, alternating between FITC, 647, and 488 labeled antibody for each AHEAD cycle to avoid selection for dye binding. anti-FLAGsuggested: (Immune Biosolutions Cat# Y00004-647, RRID:AB_2622019)Cells were collected, washed in binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 0.2% maltose) and incubated with 1 µM mouse anti-HA antibody labeled and SARS-CoV-2 RBD directly labeled with AF647. anti-HAsuggested: NoneCells were then washed and incubated with 0.5 µM goat anti-mouse AF488-labeled antibody (ThermoFisher) for 15 minutes. anti-mouse AF488-labeledsuggested: NoneExperimental Models: Cell Lines Sentences Resources To delete MET17, F102-2 cells were transformed with a linear DNA fragment encoding a G418 resistance cassette flanked on both sides by 45bp sequences homologous to the surrounding regions of MET17 (SG ID S000004294). F102-2suggested: NoneFor each nanobody-Fc fusion, 100 mL of Expi293 cells were transfected with 90-150 µg of plasmid. Expi293suggested: RRID:CVCL_D615)Mixtures were then added to HEK293T cells overexpressing human ACE2 (a gift from Dr. Huihui Mou and Dr. Michael Farzan, Scripps Research). HEK293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)Software and Algorithms Sentences Resources Data from three independent experiments were fit to a one-site model in GraphPad Prism. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)FACS selection for improved RBD binders using the improved second-generation AHEAD system After cloning the RBD-binding parent nanobodies into AHEAD (see Materials and Methods section “cloning nanobodies into AHEAD”), initial cultures (50 mL SC-HLUW) were grown to saturation and optionally passaged once or twice into 50 mL SC-HLUW at a ratio of 1:1000 to prolong diversification of the nanobodies before the first AHEAD cycle. AHEADsuggested: (AHEAD, RRID:SCR_008890)Results from OddPub: Thank you for sharing your data.
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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on pages 26, 28 and 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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