Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca 2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-goat IgG, IRDye® 800CW , LI-COR , Cat # 926-32214.

Techniques: Functional Assay, Recombinant, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Inhibition, Fluorescence, SYBR Green Assay, Activation Assay, Bicinchoninic Acid Protein Assay, In Situ, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Software

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Workflow and timeline for SARS-CoV-2 neutralizing antibodies identification The overall scheme shows three different phases for the identification of SARS-CoV-2 neutralizing antibodies (nAbs). Phase 1 consisted in the enrolment of COVID-19 patients (n = 14) from which PBMCs were isolated. Memory B cells were single-cell sorted (n = 4,277), and after 2 weeks of incubation, antibodies were screened for their binding specificity against the S protein trimer and S1/S2 domains. Once S protein-specific monoclonal antibodies (mAbs) were identified (n = 1,731) phase 2 started. All specific mAbs were tested in vitro to evaluate their neutralization activity against the authentic SARS-CoV-2 virus, and 453 nAbs were identified. nAbs showing different binding profiles on the S protein surface were selected for further functional characterization and to identify different neutralizing regions on the antigen. Phase 3 starts with the characterization of the heavy and light chain sequences of selected mAbs (n = 14) and the engineering of the Fc portion of three most promising candidates. The latter were also selected for structural analyses that allowed the identification of the neutralizing epitopes on the S protein. Finally, the most potent antibody was tested for its prophylactic and therapeutic effect in a golden Syrian hamster model of SARS-CoV-2 infection.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Isolation, Incubation, Binding Assay, In Vitro, Neutralization, Activity Assay, Virus, Functional Assay, Infection

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Gating strategy for single-cell sorting and monoclonal antibodies screening for S protein S1 + S2 subunits binding and neutralization of binding (NoB) activity, related to Figure 2 (A) Starting from top left to the right panel, the gating strategy shows: Live/Dead; Morphology; CD19 + B cells; CD19 + CD27 + IgD - ; CD19 + CD27 + IgD - IgM - ; CD19 + CD27 + IgD - IgM - S-protein + B cells. (B) The graph shows supernatants tested for binding to the SARS-CoV-2 S-protein S1 + S2 subunits. Threshold of positivity has been set as two times the value of the blank (dotted line). Darker dots represent mAbs which bind to the S1 + S2 while light yellow dots represent mAbs which do not bind. (B) The graph shows supernatants tested by NoB assay. Threshold of positivity has been set as 50% of binding neutralization (dotted line). Dark blue dots represent mAbs able to neutralize the binding between SARS-CoV-2 and receptors on Vero E6 cells, while light blue dots represent non-neutralizing mAbs.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: FACS, Binding Assay, Neutralization, Activity Assay

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Characterization and distribution of SARS-CoV-2 S protein-specific nAbs, related to Figure 2 (A) The bar graph shows the distribution of nAbs binding to different S-protein domains. In dark red, light blue and gray are shown antibodies binding to the S1-domain, S2-domain and S-protein trimer respectively. The total number (n) of antibodies tested per individual is shown on top of each bar. (B) The bar graph shows the distribution of nAbs with different neutralization potencies. nAbs were classified as weakly neutralizing (> 500 ng/mL; pale orange), medium neutralizing (100 – 500 ng/mL; orange), highly neutralizing (10 – 100 ng/mL; dark orange) and extremely neutralizing (1 – 10 ng/mL; dark red). The total number (n) of antibodies tested per individual is shown on top of each bar.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Binding Assay, Neutralization

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Functional characterization of potent SARS-CoV-2 S protein-specific nAbs (A–C) Graphs show binding curves to the S protein in its trimeric conformation, S1 domain, and S2 domain. Mean ± SD of technical triplicates are shown. Dashed lines represent the threshold of positivity. (D–F) Neutralization curves for selected antibodies were shown as percentage of viral neutralization against the authentic SARS-CoV-2 wild type (D), D614G variant (E), and the emerging variant B.1.1.7 (F). Data are representative of technical triplicates. A neutralizing COVID-19 convalescent plasma and an unrelated plasma were used as positive and negative control, respectively. (G–I) Neutralization potency of 14 selected antibodies against the authentic SARS-CoV-2 wild type (G), D614G variant (H), and the emerging variant B.1.1.7 (I). Dashed lines show different ranges of neutralization potency (500, 100, and 10 ng/mL). In all graphs, selected antibodies are shown in dark red, pink, gray, and light blue based on their ability to recognize the SARS-CoV-2 S1 RBD, S1 domain, S protein trimer only, and S2 domain, respectively.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Functional Assay, Binding Assay, Neutralization, Variant Assay, Negative Control

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Neutralization activity of selected nAbs against SARS-CoV-2, SARS-CoV, and MERS-CoV pseudotypes, related to Figure 3 (A–D) Graphs show the neutralizing activities of 14 selected nAbs with different SARS-CoV-2 S-protein binding profiles against SARS-CoV-2, SARS-CoV-2 D614G, SARS-CoV and MERS-CoV pseudotypes respectively. Dashed lines represent the threshold of positivity. Mean ± SD of technical duplicates are shown. In all graphs selected antibodies are shown in dark red, pink, gray and light blue based on their ability to recognize the SARS-CoV-2 S1-RBD, S1-domain, S-protein trimer only and S2-domain respectively.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Neutralization, Activity Assay, Protein Binding

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet: Characterization of Fc-engineered candidate nAbs, related to Figure 7 (A) the graph shows binding curves of J08, I14 and F05 MUT and WT to the FcγR2A. (B and C) graphs show binding curves of J08, I14 and F05 MUT and WT to the FcRn at pH 6.2 (B) and 7.4 (C). (D and E) Graphs show the ADNP and ADNK induced by J08, I14 and F05 MUT and WT versions; all the experiments were run as technical duplicates. In every experiment a control antibody (CR3022) and an unrelated protein were used as positive and negative control respectively. (F–H) Graphs show binding curves to the S-protein in its trimeric conformation, S1-domain and S2-domain. Mean of technical triplicates are shown. (I–K) Neutralization curves against the authentic SARS-CoV-2 wild type, the D614G variant and the B.1.1.7 emerging variant for J08-MUT, I14-MUT and F05-MUT shown in blue, green and red respectively. Data are representative of technical triplicates.

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Binding Assay, Negative Control, Neutralization, Variant Assay

Journal: Cell

Article Title: Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

doi: 10.1016/j.cell.2021.02.035

Figure Lengend Snippet:

Article Snippet: SARS Coronavirus Spike Glycoprotein (S2) , The Native Antigen Company , Cat#REC31807.

Techniques: Virus, Recombinant, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Antibody Labeling, Bicinchoninic Acid Protein Assay, Clone Assay, Random Hexamer Labeling, Plasmid Preparation, Mutagenesis, Luciferase, Software

Journal: Cell Reports

Article Title: Cancer-Specific Loss of p53 Leads to a Modulation of Myeloid and T Cell Responses

doi: 10.1016/j.celrep.2019.12.028

Figure Lengend Snippet:

Article Snippet: IRDye® 680LT Donkey anti-Rabbit , Li-Cor , Cat# 926-68023; RRID: AB_10706167.

Techniques: Functional Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software