rabbit anti ace2 antibody  (sino biological)


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    Name:
    ACE2 Angiotensin Converting Enzyme 2 Antibody Rabbit MAb
    Description:
    This antibody was obtained from a rabbit immunized with purified recombinant Rat ACE2 rR ACE2 Catalog 80031 R08H Q5EGZ1 Met1 Thr740
    Catalog Number:
    80031-R039
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    Rat
    Applications:
    IHC-P
    Immunogen:
    Recombinant Rat ACE2 protein (Catalog#80031-R08H)
    Antibody Type:
    MAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    sino biological rabbit anti ace2 antibody
    Analysis of purified plant-produced <t>ACE2-Fc</t> (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .
    This antibody was obtained from a rabbit immunized with purified recombinant Rat ACE2 rR ACE2 Catalog 80031 R08H Q5EGZ1 Met1 Thr740
    https://www.bioz.com/result/rabbit anti ace2 antibody/product/sino biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ace2 antibody - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2"

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.604663

    Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .
    Figure Legend Snippet: Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .

    Techniques Used: Purification, Produced, Staining, SDS Page, Western Blot

    Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.
    Figure Legend Snippet: Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.

    Techniques Used: Expressing, Produced, Indirect ELISA

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.
    Figure Legend Snippet: Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

    Techniques Used: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .
    Figure Legend Snippet: Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

    Techniques Used: Expressing, Plasmid Preparation, Binding Assay, Produced

    2) Product Images from "Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2"

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.604663

    Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .
    Figure Legend Snippet: Analysis of purified plant-produced ACE2-Fc (lane 1) and commercial HEK293-produced ACE2-Fc (lane 2). Coomassie-stained SDS-PAGE under reducing (A) and non-reducing conditions (B) . Western blotting analysis under reducing condition with detection using a rabbit anti-ACE2 antibody (C) and an anti-human gamma-HRP conjugated antibody (D) . Western blotting analysis under non-reducing condition probed with a rabbit anti-ACE2 antibody (E) and an anti-human gamma-HRP conjugated antibody (F) .

    Techniques Used: Purification, Produced, Staining, SDS Page, Western Blot

    Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.
    Figure Legend Snippet: Expression profiles of ACE2-Fc in N. benthamiana leaves on days 2, 4, 6, 8, and 10 after agroinfiltration. Leaf necrosis (A) Quantification of plant-produced ACE2-Fc (B) . The infiltrated leaves were collected from 3 individual plants in each day post infiltration. Data were analyzed by indirect ELISA assay using ACE2-specific antibody and presented as mean ± SD of triplicates.

    Techniques Used: Expressing, Produced, Indirect ELISA

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.
    Figure Legend Snippet: Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

    Techniques Used: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .
    Figure Legend Snippet: Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

    Techniques Used: Expressing, Plasmid Preparation, Binding Assay, Produced

    Related Articles

    Incubation:

    Article Title: Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody
    Article Snippet: For cell-entry and neutralization assays, HEK293T-ACE2 cells were seeded in 96 well plates at 50,000 cells per well. .. The next day, pseudoviral preparations normalized for p24 levels (Lenti-X™ GoStix™ Plus) were incubated with dilutions of the indicated antibodies, ACE2-mFc (SinoBiological), or media alone for 1 hr at 37°C prior to addition to cells and incubation for 48 hrs. .. Cells were then lysed and luciferase activity assessed using the ONE-Glo™ EX Luciferase Assay System (Promega) according to the manufacturer’s specifications.

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2
    Article Snippet: .. After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C. .. Then, goat anti-rabbit IgG-HRP fusion was added with the dilution of 1:2,000 in 1xPBS (BosterBio, United States) and incubated for 1 h at 37°C.

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: “Acute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques”
    Article Snippet: Sections were washed in a solution of phosphate-buffered saline and fish gelatin (PBS-FSG) and transferred to a humidified chamber. .. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with the primary antibodies (SARS-CoV-2 nucleoprotein, mouse IgG1 (Sino Biological, cat#40143-MM08); ACE2, rabbit polyclonal (Millipore, cat# HPA000288); Iba-1, rabbit polyclonal (Wako, cat# 019-19741); or pancytokeratin, rabbit polyclonal (Dako, cat#Z0622)) diluted in NGS at a concentration of 1:200 and 1:100, respectively). ..

    Article Title: The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry
    Article Snippet: After the mesh was dried for several minutes, the morphology of the exosomes was analyzed with HT7700 transmission electron microscope (Hitachi). .. For immunogold labeling, purified exosomes derived from HEK293T cells suspended with 2% PFA were placed on formvar carbon-coated nickel grids, blocked, and incubated with anti-ACE2 primary antibody (1:50 dilution, Sino Biological lnc), followed by incubation with the anti-rabbit secondary antibody conjugated with protein A-gold particles (10 nm). .. To verify the size and concentration of exosomes with NTA, purified exosomes were suspended in PBS and analyzed using a ZetaVIEW S/N 17-310 (PARTICLE METRIX).

    Blocking Assay:

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2
    Article Snippet: .. After blocking, a 1:2,000 dilution of rabbit anti-ACE2 antibody (SinoBiological, United States) in 1xPBS was added into the wells and incubated for 2 h at 37°C. .. Then, goat anti-rabbit IgG-HRP fusion was added with the dilution of 1:2,000 in 1xPBS (BosterBio, United States) and incubated for 1 h at 37°C.

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Stable Transfection:

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Expressing:

    Article Title: A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell-based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1 nM S1-mFc (Sino Biological) at 37°C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1 h at room temperature. .. The cells were washed and subsequently incubated with allophycocyanin (APC)-labeled goat anti-mouse IgG (Biolegend, catalog#405308) at 4°C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Article Title: A novel biparatopic antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy
    Article Snippet: HKE293T or CHO-S were then infected with viral supernatant and selected using puromycin. .. Cell based blocking testGradient diluted antibodies or antibody-ACE2 fusion proteins were first pre-incubated with 0.1nM S1-mFc (SinoBiological) at 37 °C overnight, followed by incubation with CHO cells stably expressing ACE2 protein (CHO-ACE2) for 1h at RT. .. The cells were washed and subsequently incubated with APC-labeled goat anti-mouse IgG (ebioscience) at 4 °C for 30 min. APC fluorescence signals were determined using a Beckman flow cytometer and results were analyzed using GraphPad7 software.

    Infection:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: INFECTION OF ACE2-HEK293 WITH SARS-COV-2 SPIKE PROTEIN PSEUDOTYPED LENTIVIRUS ACE2-HEK293s (BPS Bioscience) were cultured and transduced in opaque 96-well white plates (Corning®) with pseudotyped lentivirions displaying the SARS-CoV-2 spike glycoprotein (BPS Bioscience). .. A neutralizing monoclonal IgG antibody against the SARS-CoV-2 spike glycoprotein (CR3022, antibodies-online), ACE2 (Sino Biological), receptor-binding domain (RBD) of spike glycoprotein (Sino Biological), and SARS-BLOCK™ peptides (Ligandal) were used as inhibitors of infection. .. Infection was quantitated via bioluminescence, and toxicity was characterized via a trypan blue absorbance assay utilizing a Synergy™ H1 BioTek spectrophotometer 60h following viral transduction.

    Cell Culture:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: AUTHOR CONTRIBUTIONS AW prepared the manuscript, performed crude in silico docking of RaptorX-simulated peptides, and conceived and designed SARS-BLOCK™ peptides as well as the antibody and TCR epitope designs. .. LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2. .. AW performed spectrophotometry and trypan blue assays on the transduced cells.

    Transduction:

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)
    Article Snippet: AUTHOR CONTRIBUTIONS AW prepared the manuscript, performed crude in silico docking of RaptorX-simulated peptides, and conceived and designed SARS-BLOCK™ peptides as well as the antibody and TCR epitope designs. .. LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2. .. AW performed spectrophotometry and trypan blue assays on the transduced cells.

    Next-Generation Sequencing:

    Article Title: “Acute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques”
    Article Snippet: Sections were washed in a solution of phosphate-buffered saline and fish gelatin (PBS-FSG) and transferred to a humidified chamber. .. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with the primary antibodies (SARS-CoV-2 nucleoprotein, mouse IgG1 (Sino Biological, cat#40143-MM08); ACE2, rabbit polyclonal (Millipore, cat# HPA000288); Iba-1, rabbit polyclonal (Wako, cat# 019-19741); or pancytokeratin, rabbit polyclonal (Dako, cat#Z0622)) diluted in NGS at a concentration of 1:200 and 1:100, respectively). ..

    Concentration Assay:

    Article Title: “Acute Respiratory Distress and Cytokine Storm in Aged, SARS-CoV-2 Infected African Green Monkeys, but not in Rhesus Macaques”
    Article Snippet: Sections were washed in a solution of phosphate-buffered saline and fish gelatin (PBS-FSG) and transferred to a humidified chamber. .. Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60-minute incubation with the primary antibodies (SARS-CoV-2 nucleoprotein, mouse IgG1 (Sino Biological, cat#40143-MM08); ACE2, rabbit polyclonal (Millipore, cat# HPA000288); Iba-1, rabbit polyclonal (Wako, cat# 019-19741); or pancytokeratin, rabbit polyclonal (Dako, cat#Z0622)) diluted in NGS at a concentration of 1:200 and 1:100, respectively). ..

    Labeling:

    Article Title: The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry
    Article Snippet: After the mesh was dried for several minutes, the morphology of the exosomes was analyzed with HT7700 transmission electron microscope (Hitachi). .. For immunogold labeling, purified exosomes derived from HEK293T cells suspended with 2% PFA were placed on formvar carbon-coated nickel grids, blocked, and incubated with anti-ACE2 primary antibody (1:50 dilution, Sino Biological lnc), followed by incubation with the anti-rabbit secondary antibody conjugated with protein A-gold particles (10 nm). .. To verify the size and concentration of exosomes with NTA, purified exosomes were suspended in PBS and analyzed using a ZetaVIEW S/N 17-310 (PARTICLE METRIX).

    Purification:

    Article Title: The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry
    Article Snippet: After the mesh was dried for several minutes, the morphology of the exosomes was analyzed with HT7700 transmission electron microscope (Hitachi). .. For immunogold labeling, purified exosomes derived from HEK293T cells suspended with 2% PFA were placed on formvar carbon-coated nickel grids, blocked, and incubated with anti-ACE2 primary antibody (1:50 dilution, Sino Biological lnc), followed by incubation with the anti-rabbit secondary antibody conjugated with protein A-gold particles (10 nm). .. To verify the size and concentration of exosomes with NTA, purified exosomes were suspended in PBS and analyzed using a ZetaVIEW S/N 17-310 (PARTICLE METRIX).

    Derivative Assay:

    Article Title: The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry
    Article Snippet: After the mesh was dried for several minutes, the morphology of the exosomes was analyzed with HT7700 transmission electron microscope (Hitachi). .. For immunogold labeling, purified exosomes derived from HEK293T cells suspended with 2% PFA were placed on formvar carbon-coated nickel grids, blocked, and incubated with anti-ACE2 primary antibody (1:50 dilution, Sino Biological lnc), followed by incubation with the anti-rabbit secondary antibody conjugated with protein A-gold particles (10 nm). .. To verify the size and concentration of exosomes with NTA, purified exosomes were suspended in PBS and analyzed using a ZetaVIEW S/N 17-310 (PARTICLE METRIX).

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  • 97
    Sino Biological anti ace2 antibody rabbit polyclonal
    SARS-CoV-2 protein antigens co-express with <t>ACE2</t> in kidney tubules. a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.
    Anti Ace2 Antibody Rabbit Polyclonal, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ace2 antibody rabbit polyclonal/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ace2 antibody rabbit polyclonal - by Bioz Stars, 2021-06
    97/100 stars
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    95
    Sino Biological intracellular ace2
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of <t>ACE2‐overexpressing</t> cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Intracellular Ace2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Sino Biological ace2
    Biolayer interferometry was used to determine dissociation constants of SARS-BLOCK™ peptides associated with an IgG neutralizing antibody. Of note, Peptide 5 exhibited nonspecific binding to the sensor tip (c), preventing determination of Kd against the neutralizing antibody, and this was observed in all studies that did not utilize biotinylated substrates with biotin blocking of the sensor surface. However, we determined single-micromolar binding affinities for all other peptides with the neutralizing antibody (a,b,d). Next, we measured the dissociation constant of increasing concentrations of RBD with anti-RBD neutralizing antibody (e). Finally, 117nM RBD was mixed with increasing concentrations of <t>ACE2</t> prior to introduction to immobilized neutralizing antibody, in order to demonstrate ACE2’s inhibition of neutralizing antibody binding to the RBD (g). The IC50 of ACE2 inhibiting interaction of RBD with the neutralizing antibody is interpolated to be ∼30-35nM for ACE2 when the RBD concentration is 117nM, confirming that ACE2 binds more potently to the RBD than the neutralizing antibody does, and that soluble ACE2 can act as a potent “cloak” against neutralizing antibody recognition even at fractional molarities to SARS-CoV-2 spike RBDs.
    Ace2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SARS-CoV-2 protein antigens co-express with ACE2 in kidney tubules. a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

    Journal: Nature Communications

    Article Title: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

    doi: 10.1038/s41467-021-22781-1

    Figure Lengend Snippet: SARS-CoV-2 protein antigens co-express with ACE2 in kidney tubules. a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

    Article Snippet: Immunofluorescence double-staining For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively.

    Techniques: Expressing, Immunohistochemistry

    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Article Snippet: [ ]Commercial antibodies were used to detect intracellular ACE2 (Sino Biological, 10108‐T56) and GAPDH (Proteintech, HRP‐60004) according to the manufacturer's instructions.

    Techniques: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay

    Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p

    Article Snippet: [ ]Commercial antibodies were used to detect intracellular ACE2 (Sino Biological, 10108‐T56) and GAPDH (Proteintech, HRP‐60004) according to the manufacturer's instructions.

    Techniques: Infection, Incubation, Confocal Microscopy, Fluorescence

    Biolayer interferometry was used to determine dissociation constants of SARS-BLOCK™ peptides associated with an IgG neutralizing antibody. Of note, Peptide 5 exhibited nonspecific binding to the sensor tip (c), preventing determination of Kd against the neutralizing antibody, and this was observed in all studies that did not utilize biotinylated substrates with biotin blocking of the sensor surface. However, we determined single-micromolar binding affinities for all other peptides with the neutralizing antibody (a,b,d). Next, we measured the dissociation constant of increasing concentrations of RBD with anti-RBD neutralizing antibody (e). Finally, 117nM RBD was mixed with increasing concentrations of ACE2 prior to introduction to immobilized neutralizing antibody, in order to demonstrate ACE2’s inhibition of neutralizing antibody binding to the RBD (g). The IC50 of ACE2 inhibiting interaction of RBD with the neutralizing antibody is interpolated to be ∼30-35nM for ACE2 when the RBD concentration is 117nM, confirming that ACE2 binds more potently to the RBD than the neutralizing antibody does, and that soluble ACE2 can act as a potent “cloak” against neutralizing antibody recognition even at fractional molarities to SARS-CoV-2 spike RBDs.

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Biolayer interferometry was used to determine dissociation constants of SARS-BLOCK™ peptides associated with an IgG neutralizing antibody. Of note, Peptide 5 exhibited nonspecific binding to the sensor tip (c), preventing determination of Kd against the neutralizing antibody, and this was observed in all studies that did not utilize biotinylated substrates with biotin blocking of the sensor surface. However, we determined single-micromolar binding affinities for all other peptides with the neutralizing antibody (a,b,d). Next, we measured the dissociation constant of increasing concentrations of RBD with anti-RBD neutralizing antibody (e). Finally, 117nM RBD was mixed with increasing concentrations of ACE2 prior to introduction to immobilized neutralizing antibody, in order to demonstrate ACE2’s inhibition of neutralizing antibody binding to the RBD (g). The IC50 of ACE2 inhibiting interaction of RBD with the neutralizing antibody is interpolated to be ∼30-35nM for ACE2 when the RBD concentration is 117nM, confirming that ACE2 binds more potently to the RBD than the neutralizing antibody does, and that soluble ACE2 can act as a potent “cloak” against neutralizing antibody recognition even at fractional molarities to SARS-CoV-2 spike RBDs.

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Blocking Assay, Binding Assay, Inhibition, Concentration Assay

    Peptides simulated via RaptorX were aligned with the SARS-CoV-2 binding interface of ACE2 (ACE2 in red, with PDBePISA-predicted binding interfaces in green). Shown from left to right (top) are SARS-BLOCK™ Peptides 1 (a), 4 (b), 5 (c), and 6 (d) bound to ACE2. Of note, all peptides exhibited two mutations introducing a disulfide bond to recreate the beta sheet structure of the SARS-CoV-2 receptor binding motif (RBM). Otherwise, Peptides 1 and 4 utilized the wildtype sequence, while Peptide 5 utilized MHC-I and MHC-II epitopes, and Peptide 6 utilized a GSGSG linker (white) in one of its non-ACE2-interfacing loop regions. Peptides 4, 5 and 6 exhibited additional, proprietary modifications to their sequences to facilitate appropriate folding, while Peptide 1 lacked this modification. Taking into account the 9 possible folded states generated for each peptide, we utilized PyMOL align commands which take into account multiple potential conformations of each peptide and may serve as a basis for future studies exploring more advanced molecular dynamics approaches for relaxing and simulating intramolecular interactions at the binding interface (e). In essence, the overlay of many possible folded states represents an electron distribution cloud of possible states that can be simulated for the minimal interfacial free energy of binding, and this approach requires vastly fewer computational resources than typically required for modeling binding pockets of de novo peptides or protein-protein interfaces without existing interfacial structures.

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Peptides simulated via RaptorX were aligned with the SARS-CoV-2 binding interface of ACE2 (ACE2 in red, with PDBePISA-predicted binding interfaces in green). Shown from left to right (top) are SARS-BLOCK™ Peptides 1 (a), 4 (b), 5 (c), and 6 (d) bound to ACE2. Of note, all peptides exhibited two mutations introducing a disulfide bond to recreate the beta sheet structure of the SARS-CoV-2 receptor binding motif (RBM). Otherwise, Peptides 1 and 4 utilized the wildtype sequence, while Peptide 5 utilized MHC-I and MHC-II epitopes, and Peptide 6 utilized a GSGSG linker (white) in one of its non-ACE2-interfacing loop regions. Peptides 4, 5 and 6 exhibited additional, proprietary modifications to their sequences to facilitate appropriate folding, while Peptide 1 lacked this modification. Taking into account the 9 possible folded states generated for each peptide, we utilized PyMOL align commands which take into account multiple potential conformations of each peptide and may serve as a basis for future studies exploring more advanced molecular dynamics approaches for relaxing and simulating intramolecular interactions at the binding interface (e). In essence, the overlay of many possible folded states represents an electron distribution cloud of possible states that can be simulated for the minimal interfacial free energy of binding, and this approach requires vastly fewer computational resources than typically required for modeling binding pockets of de novo peptides or protein-protein interfaces without existing interfacial structures.

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Binding Assay, Blocking Assay, Sequencing, Modification, Generated

    Initially, an “align” command was utilized in PyMOL with SARS-CoV-1 bound to ACE2 (PDB ID 6CS2) in order to approximate the binding interface of the SWISS-MODEL simulated SARS-CoV-2 (a). Selected MHC-I and MHC-II epitope regions for inclusion in Peptide 5 are colored pink and represent P807-K835 and A1020-Y1047 in the S1 spike protein, and were further refined by IEDB immune epitope analysis (a). Next, the receptor-binding domain of the SARS-CoV-2 S1 spike protein was truncated from the larger structure in its bound state to ACE2 (b). The resulting RBD structure was run through PDBePISA to determine interacting residues, highlighted in green on ACE2 (a and b). In b, green residues on the RBD indicate predicted thermodynamically favorable interactions between ACE2 and the S1 spike protein RBD, yellow indicate predicted thermodynamically neutral and orange indicate predicted thermodynamically unfavorable interactions. Cyan residues indicate the outer bounds of amino acids used to generate SARS-BLOCK™ peptides (V433 - V511). While the predicted binding residues did not overlap completely with subsequently empirically-validated sequences, the stretches of amino acids reflected in our motifs accurately reflected binding behavior (whereby N439, Y449 , Y453, Q474, G485, N487 , Y495, Q498 , P499, and Q506 were suggested to be critical ACE2-interfacing residues by our PDBePISA simulation, while other groups’ subsequent mutagenesis studies determined that G446, Y449 , Y453, L455, F456, Y473, A475, G476, E484, F486, N487 , Y489, F490, Q493, G496, Q498 , T500, N501, G502, and Y505 are critical for binding within the stretch of S425 - Y508). 35 In sum, these residue predictions can be assessed as being precise, and accurate to within a few amino acids of actual binding behaviors—and represent a rapid and computationally minimalistic way to predict binding protein stretches without a structure when sufficiently long amino acid sequences are employed. Critically, we did not have to refine the amino acid sequences of SARS-BLOCK™ peptides to arrive at stretches of amino acids that were not empirically validated at the time of design.

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Initially, an “align” command was utilized in PyMOL with SARS-CoV-1 bound to ACE2 (PDB ID 6CS2) in order to approximate the binding interface of the SWISS-MODEL simulated SARS-CoV-2 (a). Selected MHC-I and MHC-II epitope regions for inclusion in Peptide 5 are colored pink and represent P807-K835 and A1020-Y1047 in the S1 spike protein, and were further refined by IEDB immune epitope analysis (a). Next, the receptor-binding domain of the SARS-CoV-2 S1 spike protein was truncated from the larger structure in its bound state to ACE2 (b). The resulting RBD structure was run through PDBePISA to determine interacting residues, highlighted in green on ACE2 (a and b). In b, green residues on the RBD indicate predicted thermodynamically favorable interactions between ACE2 and the S1 spike protein RBD, yellow indicate predicted thermodynamically neutral and orange indicate predicted thermodynamically unfavorable interactions. Cyan residues indicate the outer bounds of amino acids used to generate SARS-BLOCK™ peptides (V433 - V511). While the predicted binding residues did not overlap completely with subsequently empirically-validated sequences, the stretches of amino acids reflected in our motifs accurately reflected binding behavior (whereby N439, Y449 , Y453, Q474, G485, N487 , Y495, Q498 , P499, and Q506 were suggested to be critical ACE2-interfacing residues by our PDBePISA simulation, while other groups’ subsequent mutagenesis studies determined that G446, Y449 , Y453, L455, F456, Y473, A475, G476, E484, F486, N487 , Y489, F490, Q493, G496, Q498 , T500, N501, G502, and Y505 are critical for binding within the stretch of S425 - Y508). 35 In sum, these residue predictions can be assessed as being precise, and accurate to within a few amino acids of actual binding behaviors—and represent a rapid and computationally minimalistic way to predict binding protein stretches without a structure when sufficiently long amino acid sequences are employed. Critically, we did not have to refine the amino acid sequences of SARS-BLOCK™ peptides to arrive at stretches of amino acids that were not empirically validated at the time of design.

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Binding Assay, Blocking Assay, Mutagenesis

    Biolayer interferometry was used to determine dissociation constants of SARS-BLOCK™ peptides associated with dimeric ACE2, and the inhibitory effects of peptides on ACE2 binding to RBD. All peptides exhibited potent inhibition of RBD binding to ACE2 at 10μM concentrations. Peptides were associated with ACE2 at 1, 3 and 10μM concentrations until saturation was observed (a-d). After peptide binding to ACE2, we measured ACE2 association of SARS-CoV-2 RBD at 35μM in the absence of peptides (e-h). Curiously, association of ACE2 with Peptide 5 at 1uM and 3uM enhanced RBD binding, while 10uM concentrations strongly abrogated binding (g). All other peptides exhibited a dose-response-like behavior in preventing RBD binding, including at 1 and 3μM concentrations (e,g,h). Of note, peptides were not included within the final solution of 35μM RBD, meaning that this assay was more of a measure of competitive irreversible antagonism versus IC50 determination. Finally, we compared these results to RBD-biotin captured on streptavidin sensor tips, and subsequently bound to monomeric ACE2 (i).

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Biolayer interferometry was used to determine dissociation constants of SARS-BLOCK™ peptides associated with dimeric ACE2, and the inhibitory effects of peptides on ACE2 binding to RBD. All peptides exhibited potent inhibition of RBD binding to ACE2 at 10μM concentrations. Peptides were associated with ACE2 at 1, 3 and 10μM concentrations until saturation was observed (a-d). After peptide binding to ACE2, we measured ACE2 association of SARS-CoV-2 RBD at 35μM in the absence of peptides (e-h). Curiously, association of ACE2 with Peptide 5 at 1uM and 3uM enhanced RBD binding, while 10uM concentrations strongly abrogated binding (g). All other peptides exhibited a dose-response-like behavior in preventing RBD binding, including at 1 and 3μM concentrations (e,g,h). Of note, peptides were not included within the final solution of 35μM RBD, meaning that this assay was more of a measure of competitive irreversible antagonism versus IC50 determination. Finally, we compared these results to RBD-biotin captured on streptavidin sensor tips, and subsequently bound to monomeric ACE2 (i).

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Blocking Assay, Binding Assay, Inhibition

    Peptide 5 exhibited an IC50 in the sub-micromolar range and an IC95 of ∼2.22uM. Of note, in parallel, we observed virtually complete inhibition of SARS-CoV-2 spike protein pseudotyped virus infection by soluble RBD and soluble ACE2 at 0.33uM, while a SARS-CoV-2 neutralizing antibody inhibited infection to a similar extent at concentration as low as 6nM. Intriguingly, 12nM RBD enhanced infection. (*, p

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Peptide 5 exhibited an IC50 in the sub-micromolar range and an IC95 of ∼2.22uM. Of note, in parallel, we observed virtually complete inhibition of SARS-CoV-2 spike protein pseudotyped virus infection by soluble RBD and soluble ACE2 at 0.33uM, while a SARS-CoV-2 neutralizing antibody inhibited infection to a similar extent at concentration as low as 6nM. Intriguingly, 12nM RBD enhanced infection. (*, p

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Inhibition, Infection, Concentration Assay

    Importantly, with the exceptions of 20uM dose of Peptide 5 causing cell death (and leading to visible aggregation of the peptide in solution, due to poor aqueous solubility at this concentration), the addition of peptides, soluble ACE2, or soluble RBD at different concentrations did not result in statistically significant changes in cell viability in the presence of virus, ca. 50%. The SARS-CoV-2 neutralizing antibody exhibited a statistically significant (p

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Importantly, with the exceptions of 20uM dose of Peptide 5 causing cell death (and leading to visible aggregation of the peptide in solution, due to poor aqueous solubility at this concentration), the addition of peptides, soluble ACE2, or soluble RBD at different concentrations did not result in statistically significant changes in cell viability in the presence of virus, ca. 50%. The SARS-CoV-2 neutralizing antibody exhibited a statistically significant (p

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Solubility, Concentration Assay

    Peptides 1 and 4 did not block SARS-CoV-2 spike protein pseudotyped virus infection of ACE2-HEK293 cells at concentrations below 20μM, as assessed by luciferase activity 60h post-infection. Yet, both peptides 5 and 6 impeded viral infection at 6.66 micromolar, with Peptide 5 significantly exhibiting this blocking effect in the nanomolar range (80 nM and 30 nM, p

    Journal: bioRxiv

    Article Title: Peptide Antidotes to SARS-CoV-2 (COVID-19)

    doi: 10.1101/2020.08.06.238915

    Figure Lengend Snippet: Peptides 1 and 4 did not block SARS-CoV-2 spike protein pseudotyped virus infection of ACE2-HEK293 cells at concentrations below 20μM, as assessed by luciferase activity 60h post-infection. Yet, both peptides 5 and 6 impeded viral infection at 6.66 micromolar, with Peptide 5 significantly exhibiting this blocking effect in the nanomolar range (80 nM and 30 nM, p

    Article Snippet: LF performed cell culture and lentiviral transduction of HEK-ACE2 cells in the presence of peptides, antibodies, RBD, and ACE2.

    Techniques: Blocking Assay, Infection, Luciferase, Activity Assay