antibodies nabs  (Sino Biological)


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  • 93
    Name:
    Influenza H5N1 Hemagglutinin HA Neutralizing Antibody
    Description:

    Catalog Number:
    68031-H011
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    H5N1
    Applications:
    Microneutralizaiton(MN),HemagglutininInhibition(HI)
    Isotype:
    Human IgG1
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    Structured Review

    Sino Biological antibodies nabs

    https://www.bioz.com/result/antibodies nabs/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies nabs - by Bioz Stars, 2021-04
    93/100 stars

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    Filtration:

    Article Title: Essential Role for Autophagy in the Maintenance of Immunological Memory Against Influenza Infection
    Article Snippet: Splenocytes or bone marrow cells (1–5×105/well) were then added to the plates and incubated at 37 °C for 5 h. The cells were lysed with H2 O and the wells were probed with HRP-conjugated goat anti-mouse IgG1 or anti-mouse IgM (Southern Biotechnology), followed by development with 3-amino-9-ethylcarbzole (Sigma). .. To detect HA-specific antibody forming cells, the MultiScreen Filtration plates were coated with 10 μg ml−1 H3N2 HA (Sino Biological). .. Splenocytes or lung cells (1–5×105 /well) were then added to the plates and incubated at 37 °C for 5 h. The cells were lysed with H2O and the wells were probed with HRP-conjugated goat anti-mouse IgG or anti-mouse IgA (Southern Biotechnology) and developed as above.

    Neutralization:

    Article Title: COVID-19 Severity Correlates with Weaker T-Cell Immunity, Hypercytokinemia, and Lung Epithelium Injury
    Article Snippet: Cytokines were measured by using Cytometric Bead Array kits (BD Bioscience). .. Focus reduction neutralization test was performed to evaluate the levels of neutralizing antibodies (nAbs) using Vero E6 cells infected with SARS-CoV-2 and rabbit anti–SARS-CoV-2 nucleocapsid protein polyclonal antibody (Sino Biological). .. The foci were visualized by TrueBlue reagent and counted with an ELISPOT reader (CTL S6 Ultra).

    Infection:

    Article Title: COVID-19 Severity Correlates with Weaker T-Cell Immunity, Hypercytokinemia, and Lung Epithelium Injury
    Article Snippet: Cytokines were measured by using Cytometric Bead Array kits (BD Bioscience). .. Focus reduction neutralization test was performed to evaluate the levels of neutralizing antibodies (nAbs) using Vero E6 cells infected with SARS-CoV-2 and rabbit anti–SARS-CoV-2 nucleocapsid protein polyclonal antibody (Sino Biological). .. The foci were visualized by TrueBlue reagent and counted with an ELISPOT reader (CTL S6 Ultra).

    other:

    Article Title: TRIB3 Interacts With β-Catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis.
    Article Snippet: Cell lysates were immuno-precipitated with anti–human influenza hemagglutinin antibody and Protein G Sepharose beads overnight.

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  • 95
    Sino Biological anti s2
    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using <t>anti-S2</t> Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P
    Anti S2, 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
    https://www.bioz.com/result/anti s2/product/Sino Biological
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti s2 - by Bioz Stars, 2021-04
    95/100 stars
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    93
    Sino Biological egf antibody
    Physicochemical properties of <t>HGF</t> targeting peptide a. K D of HGP-1 binding to HGF was determined by SPR technique. b. The assessment of binding competition between various proteins and HGF by fluorescence-based ELISA assay post 1.5-hour incubation. Proteins at the concentrations of 0.05 nM, 0.5 nM, 5 nM and 50 nM mixed with 10 μM FITC-labeled HGP-1 were the liquid phase ( n = 5). c. The binding activity between HGP-1 to HGF and <t>EGF</t> were measured by fluorescence-based direct ELISA assay post 1.5-hour incubation. HGP-1 at the concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM were used ( n = 3). Values were mean ± SEM.
    Egf Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egf antibody/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egf antibody - by Bioz Stars, 2021-04
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    99
    Sino Biological sars cov 2 2019 ncov nucleocapsid antibody rabbit pab
    A wide range of ACE2 orthologs support binding to RBD proteins of <t>SARS-CoV-2</t> and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.
    Sars Cov 2 2019 Ncov Nucleocapsid Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 2019 ncov nucleocapsid antibody rabbit pab/product/Sino Biological
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov nucleocapsid antibody rabbit pab - by Bioz Stars, 2021-04
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    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Journal: bioRxiv

    Article Title: Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration

    doi: 10.1101/2020.10.15.339838

    Figure Lengend Snippet: N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Article Snippet: Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc (Jackson), anti-RBD (Sino Biologicals), anti-S2 (Sino Biologicals) and anti-ACE2 (R & D Systems) pAbs.

    Techniques: Modification, Expressing, Mutagenesis, Produced, Generated, Titration, Infection, Western Blot

    Physicochemical properties of HGF targeting peptide a. K D of HGP-1 binding to HGF was determined by SPR technique. b. The assessment of binding competition between various proteins and HGF by fluorescence-based ELISA assay post 1.5-hour incubation. Proteins at the concentrations of 0.05 nM, 0.5 nM, 5 nM and 50 nM mixed with 10 μM FITC-labeled HGP-1 were the liquid phase ( n = 5). c. The binding activity between HGP-1 to HGF and EGF were measured by fluorescence-based direct ELISA assay post 1.5-hour incubation. HGP-1 at the concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM were used ( n = 3). Values were mean ± SEM.

    Journal: Oncotarget

    Article Title: The HGF inhibitory peptide HGP-1 displays promising in vitro and in vivo efficacy for targeted cancer therapy

    doi:

    Figure Lengend Snippet: Physicochemical properties of HGF targeting peptide a. K D of HGP-1 binding to HGF was determined by SPR technique. b. The assessment of binding competition between various proteins and HGF by fluorescence-based ELISA assay post 1.5-hour incubation. Proteins at the concentrations of 0.05 nM, 0.5 nM, 5 nM and 50 nM mixed with 10 μM FITC-labeled HGP-1 were the liquid phase ( n = 5). c. The binding activity between HGP-1 to HGF and EGF were measured by fluorescence-based direct ELISA assay post 1.5-hour incubation. HGP-1 at the concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM were used ( n = 3). Values were mean ± SEM.

    Article Snippet: Reagents and cell culture Recombinant human full length HGF (10463- NHAC-A), VEGF-165 (11066-HNAB), Protein G (13103-PNAE), Biotinylated recombinant human full length HGF (10463-HNAC-B), HGF antibody (10463-RP01-B), MET antibody (10692-MM02), EGF antibody (0605-R008) were purchased from Sino Biological Inc. Recombinant human EGF (H6000–10), bFGF (H3000–10) were obtained from Beijing Wishbiotechnology Co., Ltd. Dynabeads MyOne streptavidin C1 magnetic beads (#65002) and Streptavidin Phycoerythrin Conjugates (S-866) were obtained from Invitrogen.

    Techniques: Binding Assay, SPR Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Activity Assay, Direct ELISA

    A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: A wide range of ACE2 orthologs support binding to RBD proteins of SARS-CoV-2 and three related coronaviruses. (A) 293T cells were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then stained with an RBD-mouse IgG2 Fc fusion protein of SARS-CoV-2 WHU01, Pangolin-CoV-2020, Bat-CoV RaTG13, or SARS-CoV BJ01, followed by staining with an Alexa 488-goat anti-mouse IgG secondary antibody. RBD-ACE2 binding was detected using flow cytometry. (B) Percentages of cells positive for RBD binding in panel A are presented as a heatmap according to the indicated color code. (C) Expression levels of the indicated ACE2 orthologs were detected using Western blotting. The data shown are representative of two independent experiments performed by two different people with similar results.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Binding Assay, Transfection, Expressing, Staining, Flow Cytometry, Western Blot

    A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: A wide range of ACE2 orthologs support cell entry of SARS-CoV-2 and three related coronaviruses. (A to F) 293T cells in 96-well plates were transfected with adjusted amounts of the indicated ACE2-ortholog plasmids to have similar expression levels of the ACE2 ortholog proteins. Cells were then infected with retrovirus-based luciferase reporter pseudoviral particles (pp) enveloped with the indicated spike proteins. ACE2 ortholog-mediated viral entry was measured by luciferase reporter expression at 48 h (A to D and F) or 60 h (E) postinfection. (G) The relative infection (%) values for each ACE2 ortholog-mediated viral entry shown in panels A to F were independently calculated against the highest expression values of the same pseudotype panel and are presented as a heatmap according to the indicated color code. (H) 293T cells expressing ACE2 orthologs of the indicated species were infected with SARS-CoV-2 live virus at 800 TCID 50 . Cells were then fixed and stained with rabbit anti-SARS-CoV-2 nucleocapsid (NP) polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP, and blue indicates cell nuclei. Scale bars, 50 μm. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to F represent the means ± the SD of four biological replicates.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Transfection, Expressing, Infection, Luciferase, Staining, Fluorescence, Microscopy

    The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: The 740-D30E variant of ACE2-Ig broadly neutralizes entry of SARS-CoV-2, SARS-CoV, Pangolin-CoV-2020 and Bat-CoV RaTG13. (A to D) Human ACE2-expressing 293T were infected with the indicated pseudotypes in the presence of an Fc fusion protein, F10-scFv (gray), ACE2 740-wt (blue), or ACE2 740-D30E (red). Viral entry was measured by luciferase reporter expression at 48 h (A, B, and D) or 60 h (C) postinfection, and the percent infection (Infection%) values were calculated. Note that the D30E mutation on the ACE2-Ig protein improved the protein’s neutralization activity against SARS-CoV-2 (A) and RaTG13 (C) but not Pangolin-CoV-2020 (B) or SARS-CoV (D). The dashed line in panels C and D indicates the background luciferase signals detected in the pseudovirus-infected parental 293T cells. (E) Human ACE2 residue D30 forms a salt bridge with the SARS-CoV-2 RBD residue K417 (PDB accession no. 6M0J ). SARS-CoV-2 and RaTG13 have a K417 residue at their spike proteins, while Pangolin-CoV has an R417 residue and SARS-CoV has a V417 residue at their spike proteins, respectively. Thus, a stabilized salt bridge interaction between E30 of the ACE2-Ig protein and K417 of the virus spike protein is likely responsible for the D30E mutation-mediated neutralization enhancement. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels A to D represent the means ± the SD of three or four biological replicates.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Variant Assay, Expressing, Infection, Luciferase, Mutagenesis, Neutralization, Activity Assay

    Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: Recombinant RBD-Ig and ACE2-Ig variants efficiently block SARS-CoV-2 entry. (A) Diagrams of RBD-Ig and ACE2-Ig fusion proteins used in the following studies. (B and C) ACE2-expressing 293T cells were infected with SARS-CoV-2 spike-pseudotyped retrovirus (pp) in the presence of purified recombinant RBD-Ig (B) and ACE2-Ig (C) fusion proteins at the indicated concentrations. An Fc fusion protein of an anti-influenza HA antibody, F10-scFv, was used as a control protein here. Viral entry was measured by the luciferase reporter at 48 h postinfection. Luminescence values observed at each concentration were divided by the values observed at concentration zero to calculate the percent infection (Infection%) values. Note that all the 740-version variants showed significantly better potency than the 615-version variants (two-tailed two-sample t test, P

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Recombinant, Blocking Assay, Expressing, Infection, Purification, Luciferase, Concentration Assay, Two Tailed Test

    SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: SARS-CoV-2 and ACE2 contact residues are conserved among four SARS-like viruses and 16 ACE2 orthologs, respectively. (A) Interactions between the SARS-CoV-2 receptor binding domain (RBD, red) and ACE2 (blue) involve a large number of contact residues (PDB accession no. 6M0J ). RBD residues

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Binding Assay

    A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.

    Journal: Journal of Virology

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig

    doi: 10.1128/JVI.01283-20

    Figure Lengend Snippet: A further improved ACE2-Ig variant with an antibody-like configuration potently neutralizes SARS-CoV-2 live virus. (A) Diagrams of ACE2-Ig variants characterized in the following studies. CH1, IgG heavy-chain constant region 1; CL, human antibody kappa light-chain constant region. (B and C) Human ACE2-expressing 293T (B) or HeLa (C) cells were infected with SARS-CoV-2 pseudotype in the presence of the indicated human IgG1 Fc fusion proteins at the indicated concentrations. An anti-HIV antibody b12 was used as a human IgG1 control. Viral entry was measured by luciferase reporter expression at 48 h postinfection, and the percent infection (Infection%) values were calculated. Estimated IC 50 and IC 90 values for each protein are directly derived from the curves and are shown to the right of the figures. (D) Human ACE2-expressing HeLa cells were infected with SARS-CoV-2 live virus at 800 TCID 50 in the presence of the b12 control protein, ACE2-Ig-v1, or ACE2-Ig-v3 at the indicated concentrations. Cells were then fixed and stained with rabbit anti-SARS-CoV-2 NP polyclonal antibody for fluorescence microscopy at 24 h postinfection. Red indicates SARS-CoV-2 NP and blue indicates cell nuclei. Scale bars, 200 μm. Note that ACE2-Ig-v3 at 0.8 μg/ml (1.85 nM) completely abolished viral NP signal. The data shown are representative of two or three experiments independently performed by two different people with similar results, and data points in panels B and C represent the means ± the SD of three biological replicates.

    Article Snippet: The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus).

    Techniques: Variant Assay, Expressing, Infection, Luciferase, Derivative Assay, Staining, Fluorescence, Microscopy