Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: Neutralization of SARS-CoV-2 infection and RBD binding to ACE2 (A) Indicated hmAbs were incubated with live SARS-CoV-2 (100 PFU/well) 1 h before (pre-treatment) or 1 h after (post-treatment) addition to Vero E6 cells. hmAbs were tested in quadruplicate cultures and NT 50 and upper 95% confidence interval (CI) indicated. (B) Representative titration curve of 1212C2 hmAb presented, mean and standard error presented. (C) Binding of indicated hmAb to SARS-CoV-2 or mock-infected Vero E6 cells measured by immunofluorescence; scale bar, 100 μm. (D) Indicated hmAb was incubated as single replicate with recombinant biotinylated RBD protein before incubation with HEK293-ACE2 cells measured by flow cytometry. Plot gated on 7-aminoactinomycin (7AAD)-ACE2 + cells.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Neutralization, Infection, Binding Assay, Incubation, Titration, Immunofluorescence, Recombinant, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet: SPR epitope mapping (A) Representative sensorgram from the SPR competition assays used to subset hmAbs into distinct RBD binding epitopes. For each assay, a series of hmAbs were sequentially injected over immobilized SARS-CoV-2 RBD. In this example, 1212C2 was injected first, followed by a second injection of 1212C2, 2 injections of 1206D1, and the last injection was CR3022. (B) Summary of all epitope mapping data, in which each block (first experiment from A in the red box) with a bold hmAb at the top represents a different experiment (10 experiments total). The bold hmAb is the “first” hmAb injected. The percentage of binding (100 = 100% binding and 0 = 0% binding) of subsequent hmAbs was recorded. mAbs were considered to have a different epitope (denoted by a distinct color) if they exhibited binding levels >30% in the presence of other mAbs. Thus, in the first experiment, CR3022 is defined as a new epitope (cyan), distinct from 1212C2. (C) Schematic diagram of NmAb RBD epitopes defined in the mapping experiment. Five major epitopes (A–E) were identified, where the E epitope overlaps with control mAb CR3022 (cyan, epitope F). Four of the 5 epitopes (A–D) are located within the ACE2 binding site (purple), and all of the NmAbs are blocked by the 1212C2 epitope A (yellow). NmAbs with epitopes similar to B (orange) and C (green) are defined as B’ (light orange) and C’ (light green), respectively. The 1212C2 epitope A (yellow) blocks the binding of all NmAbs, with the exception of 1215B11, which occupies epitope E. Epitopes B and C are also blocked by epitope A NmAbs, but exhibit limited competition with each other.

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Binding Assay, Injection, Blocking Assay, Control

Journal: Cell Reports Medicine

Article Title: Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters

doi: 10.1016/j.xcrm.2021.100218

Figure Lengend Snippet:

Article Snippet: Cryopreserved cells were thawed and blocked with 0.5 μg anti-ACE2 antibody (AF933-SP, R&D System) for 5 min at room temperature and then stained for flow cytometry similar as previously described, using anti- CD19-APC-Cy7 (SJ25C1, BD Biosciences), HIV gp140-AlexaFluor647, SARS-CoV-2 RBD-BV421, CD3-BV510 (OKT3, Biolegend), CD4-BV510 (HI30, Biolegend), IgD-FITC (IA6-2, BD Biosciences), CD27-PE (CLB-27/1,Life Technologies), Annexin V-PerCP-Cy5.5 (Biolegend), and Live/Dead aqua (Molecular Probes).

Techniques: Synthesized, Expressing, Virus, Recombinant, Plasmid Preparation, Binding Assay, Saline, cDNA Synthesis, Transfection, Gel Extraction, Lysis, Luciferase, Software

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 1 In‐house and commercial ACE2 enzymatic immunoassay (EIA) results of pre‐COVID‐19 donor control sera, COVID‐19 convalescent patient, and vaccine recipient sera. (A, B) IgM EIA results of COVID‐19 convalescent sera classified based on severity. (C, D) IgG EIA results of COVID‐19 convalescent sera classified based on severity. (E, F) IgG EIA results of COVID‐19 vaccine recipients based on type of vaccine. Bars represent median and interquartile range. Intergroup comparisons of medians were performed using Dunn's multiple comparisons test. Ns: not significant; *p ≤0.05; ***p ≤0.001; ****p ≤0.0001. ACE2, angiotensin‐converting enzyme 2; COVID‐19, coronavirus disease 2019.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay, Control

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 2 Correlations between ACE2 IgG enzymatic immunoassay optical densities (OD) and surrogate neutralizing antibody levels of CoronaVac (A, B) and Comirnaty (C, D) cohorts using commercial and in‐house ACE2 peptides. Strength of correlation was assessed using Spearman's rank correlation. ACE2, angiotensin‐converting enzyme 2.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques: Enzyme Immunoassay

Journal: Journal of medical virology

Article Title: Autoantibodies against angiotensin-converting enzyme 2 (ACE2) after COVID-19 infection or vaccination.

doi: 10.1002/jmv.29313

Figure Lengend Snippet: FIGURE 3 Trends of ACE2 IgG optical densities (ODs) using in‐house (A) and commercial (B) peptides for vaccine recipients testing positive at Day 56 post‐first dose. Each line represents trend for individual recipients. SNV020, SNV027, and SNV058 are CoronaVac recipients. BNT007, BNT012, BNT032, BNT081, BNT090, and BNT092 are Comirnaty recipients. The second timepoint is either Day 21 (for Comirnaty recipients) or Day 28 (for CoronaVac recipients). ACE2, angiotensin‐converting enzyme 2.

Article Snippet: In addition, we expressed human ACE2 (Ser19‐Arg708) in‐house using a baculovirus insect cell system as described previously.18 Both commercial and in‐house ACE2 peptides were characterized using sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blot analysis using a monoclonal antibody against ACE2 (R&D Systems; Cat#:AF933).

Techniques:

Journal: eLife

Article Title: ACE2: Evidence of role as entry receptor for SARS-CoV-2 and implications in comorbidities

doi: 10.7554/eLife.61390

Figure Lengend Snippet: Available validation data for the antibodies used in the studies described in this review in accordance to the pillars defined by the Antibodypedia validation initiative ( Uhlen et al., 2016 ). Provider refers to the information found in the website of the company. IB: Immunoblot. IHC: Immunohistochemistry. IF: Immunofluorescence. Enhanced validation, Supportive validation, No data available, + Positive detection, +/- Weak detection, - Absence of detection.

Article Snippet: AF933 Immunogen: 18-74aa , IB , , + Ovary, testis and kidney , + Airway and distal lung, ALI-cultured airway epithelial cells (correlation with mRNA levels), Calu-3 and Caco-2 cells ( ; ) - A549 (expected), Huh-7 cells ( ; ) .

Techniques: Biomarker Discovery, Western Blot, Immunohistochemistry, Immunofluorescence, Immunohistochemistry-IF, Transfection, Staining