sars cov 2 rbd  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD rFc Recombinant Protein COVID 19 Spike RBD Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of rabbit IgG1 at the C terminus
    Catalog Number:
    40592-V31H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological sars cov 2 rbd
    Concentration-dependent inhibition of <t>SARS-CoV-2</t> RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of rabbit IgG1 at the C terminus
    https://www.bioz.com/result/sars cov 2 rbd/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19"

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

    Journal: bioRxiv

    doi: 10.1101/2020.08.29.273441

    Concentration-dependent inhibition of SARS-CoV-2 RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2 RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.

    Techniques Used: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay

    Concentration-response curves for binding of SARS-CoV-2 spike protein S1 and RBD to ACE2 in our ELISA-based assay format. Data obtained with Fc-conjugated ACE2 coated on the plate and His-tagged S1 or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).
    Figure Legend Snippet: Concentration-response curves for binding of SARS-CoV-2 spike protein S1 and RBD to ACE2 in our ELISA-based assay format. Data obtained with Fc-conjugated ACE2 coated on the plate and His-tagged S1 or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

    Techniques Used: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Inhibitory effect of selected compounds on SARS-CoV-2 RBD binding to hACE2 in our screening assay. Percent inhibition values obtained at 5 μM concentration shown normalized to control (100%). Erythrosine B, a known promiscuous SMI of PPIs ( Ganesan et al., 2011 ) and sunset yellow FCF (FD C yellow no. 6), a food colorant likely to be inactive, were included as positive and negative controls, respectively. Chemical structures are shown for comparison purposes.
    Figure Legend Snippet: Inhibitory effect of selected compounds on SARS-CoV-2 RBD binding to hACE2 in our screening assay. Percent inhibition values obtained at 5 μM concentration shown normalized to control (100%). Erythrosine B, a known promiscuous SMI of PPIs ( Ganesan et al., 2011 ) and sunset yellow FCF (FD C yellow no. 6), a food colorant likely to be inactive, were included as positive and negative controls, respectively. Chemical structures are shown for comparison purposes.

    Techniques Used: Binding Assay, Screening Assay, Inhibition, Concentration Assay

    2) Product Images from "Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens"

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens

    Journal: JCI Insight

    doi: 10.1172/jci.insight.139042

    Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A and B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C and D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A and B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C and D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

    Techniques Used: In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A – D ; 20 μm in inset of E ; and 10 μm in E .
    Figure Legend Snippet: Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A – D ; 20 μm in inset of E ; and 10 μm in E .

    Techniques Used: Immunohistochemistry, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bars: 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bars: 20 μm.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Infection, Staining

    Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    Figure Legend Snippet: Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded

    3) Product Images from "CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation"

    Article Title: CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation

    Journal: bioRxiv

    doi: 10.1101/2020.07.24.220715

    Detection of IFN-γ secreting cells from mice immunised with SARS-CoV-2 vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/mL or 0.5 μg/mL per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/106 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. *** p ≤ 0.001, **** p ≤ 0.0001.
    Figure Legend Snippet: Detection of IFN-γ secreting cells from mice immunised with SARS-CoV-2 vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/mL or 0.5 μg/mL per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/106 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. *** p ≤ 0.001, **** p ≤ 0.0001.

    Techniques Used: Mouse Assay, Incubation, Activity Assay

    Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. C Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in panels (B) and (C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads).
    Figure Legend Snippet: Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. C Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in panels (B) and (C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads).

    Techniques Used: Fluorescence, Binding Assay, Magnetic Beads, Negative Control

    4) Product Images from "Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens"

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens

    Journal: JCI Insight

    doi: 10.1172/jci.insight.139042

    Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A and B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C and D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A and B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C and D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

    Techniques Used: In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A – D ; 20 μm in inset of E ; and 10 μm in E .
    Figure Legend Snippet: Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A – D ; 20 μm in inset of E ; and 10 μm in E .

    Techniques Used: Immunohistochemistry, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bars: 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bars: 20 μm.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Infection, Staining

    Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    Figure Legend Snippet: Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded

    5) Product Images from "Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens"

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens

    Journal: JCI Insight

    doi: 10.1172/jci.insight.139042

    Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A and B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C and D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 RNA by ISH in FFPE cell pellets. ( A and B ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 1 in infected FFPE cell pellets ( B ), but not in uninfected control FFPE cell pellets ( A ). ( C and D ) SARS-CoV-2 positive-sense RNA can be detected by ISH using positive-sense RNA probe 2 in infected FFPE cell pellets ( D ), but not in uninfected control FFPE cell pellets ( C ). ( E and F ) SARS-CoV-2 negative-sense RNA can be detected by ISH using negative-sense RNA probe 1 in infected FFPE cell pellets ( E ), but not in uninfected control FFPE cell pellets ( F ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

    Techniques Used: In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A – D ; 20 μm in inset of E ; and 10 μm in E .
    Figure Legend Snippet: Detection of SARS-CoV-2 antigens by IHC and IFA in FFPE cell pellets. ( A and B ) In comparison to uninfected control FFPE cell pellets ( A and C ), SARS-CoV-2 S (brown, B ) and SARS-CoV-2 NP (brown, D ) can be detected in FFPE SARS-CoV-2–infected cell pellets. Nuclei are stained blue (hematoxylin). ( E ) Immunofluorescence staining to detect SARS-CoV-2 S (green) and NP (red) in FFPE SARS-CoV-2-infected cell pellets. Inset: Uninfected control FFPE cell pellets. Nuclei are stained blue (DAPI). Scale bars: 50 μm in A – D ; 20 μm in inset of E ; and 10 μm in E .

    Techniques Used: Immunohistochemistry, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Infection, Staining

    Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bars: 20 μm.
    Figure Legend Snippet: Detection of SARS-CoV-2 replication in FFPE cells using mFISH. ( A and B ) Compared with uninfected control ( A ), SARS-CoV-2 negative-sense RNA (green), a replicative intermediate that indicates viral replication, can be detected in infected FFPE cell pellets in addition to positive-sense (red) RNA ( B ). Nuclei are stained blue (DAPI). Scale bars: 20 μm.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Infection, Staining

    Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.
    Figure Legend Snippet: Dual staining to detect SARS-CoV-2 antigen and RNA in the same FFPE section. ( A and B ) Compared with uninfected control FFPE cell pellets ( A ), SARS-CoV-2 S (brown) and positive-sense RNA (red) were detected in the same section ( B ). Nuclei are stained blue (hematoxylin). Scale bars: 50 μm.

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded

    6) Product Images from "Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19"

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19

    Journal: bioRxiv

    doi: 10.1101/2020.06.18.158196

    A] Cell viability after incubation of Vero cells with imatinib for either 1 or 8 hours. and B] SARS-CoV-2 neutralization profile post 1- and 8-hours exposure to imatinib. Inhibition of VSV pseudoparticles for SARS-CoV, SARS-CoV-2, MERS-CoV and VSV(control) after incubation with imatinib in C] Vero cells and D] Vero-TMPRSS2 cells. The red arrow indicates the concentration where no toxicity was observed microscopically anymore (15 nM). E] The association and dissociation curves obtained by BLI reflecting the binding of imatinib (0.78 to 6.25 µM) to immobilized SARS-CoV-2 RBD protein. Data fitted using the 1:1 binding model are shown in black.
    Figure Legend Snippet: A] Cell viability after incubation of Vero cells with imatinib for either 1 or 8 hours. and B] SARS-CoV-2 neutralization profile post 1- and 8-hours exposure to imatinib. Inhibition of VSV pseudoparticles for SARS-CoV, SARS-CoV-2, MERS-CoV and VSV(control) after incubation with imatinib in C] Vero cells and D] Vero-TMPRSS2 cells. The red arrow indicates the concentration where no toxicity was observed microscopically anymore (15 nM). E] The association and dissociation curves obtained by BLI reflecting the binding of imatinib (0.78 to 6.25 µM) to immobilized SARS-CoV-2 RBD protein. Data fitted using the 1:1 binding model are shown in black.

    Techniques Used: Incubation, Neutralization, Inhibition, Concentration Assay, Binding Assay

    A] Docked poses of the selected compounds at the receptor-binding domain of SARS-CoV-2 spike protein (inset: conformation of imatinib from molecular dynamics simulations showing important interactions with the receptor at the active site). B] MM-GBSA binding free energies for the selected compounds with negative control DMSO. Error bars indicate standard deviations for sampling from a whole simulation. Tyrosine kinase inhibitors ponatinib and imatinib displayed a high affinity to the RBD of the spike protein.
    Figure Legend Snippet: A] Docked poses of the selected compounds at the receptor-binding domain of SARS-CoV-2 spike protein (inset: conformation of imatinib from molecular dynamics simulations showing important interactions with the receptor at the active site). B] MM-GBSA binding free energies for the selected compounds with negative control DMSO. Error bars indicate standard deviations for sampling from a whole simulation. Tyrosine kinase inhibitors ponatinib and imatinib displayed a high affinity to the RBD of the spike protein.

    Techniques Used: Binding Assay, Negative Control, Sampling

    Related Articles

    Infection:

    Article Title: CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation
    Article Snippet: .. SARS-CoV-2 is highly transmissible during both the pre-symptomatic and acute symptomatic phases and the infection fatality rate has been reported as high as 3.4% . .. COVID-19 often develops into severe illness, including pneumonia.

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: .. To evaluate whether these 6 antibodies can recognize SARS-CoV-2 in FFPE specimens, we performed IHC on FFPE pellets of Vero 76 cells infected with SARS-CoV-2. .. We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV NP (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets ( ).

    other:

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19
    Article Snippet: The plated concentrations of ACE2 receptor were 1.0 μg/mL for SARS-CoV-2 RBD and 2.0 μg/mL for SARS-CoV-2 S1.

    Immunohistochemistry:

    Article Title: Placental SARS-CoV-2 in a patient with mild COVID-19 disease
    Article Snippet: Immunohistochemistry (IHC) staining of SARS-CoV-2 virus in a COVID-19 negative patient, delivery prior to the COVID-19 outbreak (Figure 2a). .. IHC of SARS-CoV-2 from three placental sections (2b: under umbilical cord, 2c: central placental disc, 2d-e: peripheral placental disc at 20x and 40x). .. IHC of cytokeratin-7 (CK-7) marker in control ferret nasal turbinate tissue (Figure 2f).

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: .. To evaluate whether these 6 antibodies can recognize SARS-CoV-2 in FFPE specimens, we performed IHC on FFPE pellets of Vero 76 cells infected with SARS-CoV-2. .. We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV NP (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets ( ).

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: We also identify 2 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S and RNA in the same FFPE section. .. Identification of antibodies suitable for detection of SARS-CoV-2 by IHC and IFA in FFPE specimens.). ..

    Titration:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Microneutralization Assay:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Expressing:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Cell Culture:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Concentration Assay:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Binding Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: The number of infected cells per well were counted using the ImageQuant TL software. .. BLIThe binding kinetics of imatinib on SARS-CoV-2 RBD protein were studied using a BLItz® system (FortéBio). .. Experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1X Kinetics Buffer (FortéBio), 5% anhydrous dimethyl sulfoxide (DMSO; Sigma Aldrich).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: .. To evaluate whether these 6 antibodies can recognize SARS-CoV-2 in FFPE specimens, we performed IHC on FFPE pellets of Vero 76 cells infected with SARS-CoV-2. .. We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV NP (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets ( ).

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: We also identify 2 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S and RNA in the same FFPE section. .. Identification of antibodies suitable for detection of SARS-CoV-2 by IHC and IFA in FFPE specimens.). ..

    Immunofluorescence:

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: We also identify 2 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S and RNA in the same FFPE section. .. Identification of antibodies suitable for detection of SARS-CoV-2 by IHC and IFA in FFPE specimens.). ..

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    Sino Biological sars cov 2 rbd
    Concentration-dependent inhibition of <t>SARS-CoV-2</t> RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.
    Sars Cov 2 Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 2 rbd - by Bioz Stars, 2021-04
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    98
    Sino Biological anti rbd
    S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient <t>293T-hACE2-RBD</t> and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p
    Anti Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 1 article reviews
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    Concentration-dependent inhibition of SARS-CoV-2 RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.

    Journal: bioRxiv

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

    doi: 10.1101/2020.08.29.273441

    Figure Lengend Snippet: Concentration-dependent inhibition of SARS-CoV-2 RBD binding to ACE2 by selected compounds. Concentration-response curves obtained in ELISA-type assay with Fc-conjugated ACE2 coated on the plate (1 μg/mL) and His-tagged RBD (0.5 μg/mL) added and amount bound in the presence of increasing concentrations of test compounds detected. As before, erythrosine B (ErB) and sunset yellow FCF (SY(FD C#6)) were included as positive and negative controls, respectively. Data (mean ± SD for two experiments in duplicates) were normalized and fitted with standard inhibition curves; obtained IC 50 values are shown at right.

    Article Snippet: The plated concentrations of ACE2 receptor were 1.0 μg/mL for SARS-CoV-2 RBD and 2.0 μg/mL for SARS-CoV-2 S1.

    Techniques: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay

    Concentration-response curves for binding of SARS-CoV-2 spike protein S1 and RBD to ACE2 in our ELISA-based assay format. Data obtained with Fc-conjugated ACE2 coated on the plate and His-tagged S1 or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

    Journal: bioRxiv

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

    doi: 10.1101/2020.08.29.273441

    Figure Lengend Snippet: Concentration-response curves for binding of SARS-CoV-2 spike protein S1 and RBD to ACE2 in our ELISA-based assay format. Data obtained with Fc-conjugated ACE2 coated on the plate and His-tagged S1 or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

    Article Snippet: The plated concentrations of ACE2 receptor were 1.0 μg/mL for SARS-CoV-2 RBD and 2.0 μg/mL for SARS-CoV-2 S1.

    Techniques: Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Inhibitory effect of selected compounds on SARS-CoV-2 RBD binding to hACE2 in our screening assay. Percent inhibition values obtained at 5 μM concentration shown normalized to control (100%). Erythrosine B, a known promiscuous SMI of PPIs ( Ganesan et al., 2011 ) and sunset yellow FCF (FD C yellow no. 6), a food colorant likely to be inactive, were included as positive and negative controls, respectively. Chemical structures are shown for comparison purposes.

    Journal: bioRxiv

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

    doi: 10.1101/2020.08.29.273441

    Figure Lengend Snippet: Inhibitory effect of selected compounds on SARS-CoV-2 RBD binding to hACE2 in our screening assay. Percent inhibition values obtained at 5 μM concentration shown normalized to control (100%). Erythrosine B, a known promiscuous SMI of PPIs ( Ganesan et al., 2011 ) and sunset yellow FCF (FD C yellow no. 6), a food colorant likely to be inactive, were included as positive and negative controls, respectively. Chemical structures are shown for comparison purposes.

    Article Snippet: The plated concentrations of ACE2 receptor were 1.0 μg/mL for SARS-CoV-2 RBD and 2.0 μg/mL for SARS-CoV-2 S1.

    Techniques: Binding Assay, Screening Assay, Inhibition, Concentration Assay

    S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Article Snippet: The presence of the SARS-CoV-2 pseudovirus was further confirmed by flow cytometry, transfected 293T-hACE2 cells were stained with primary anti-RBD followed by goat anti-rabbit fluorophore-conjugated secondary antibody.

    Techniques: Binding Assay, Expressing, Activity Assay, Release Assay, Labeling

    Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Article Snippet: The presence of the SARS-CoV-2 pseudovirus was further confirmed by flow cytometry, transfected 293T-hACE2 cells were stained with primary anti-RBD followed by goat anti-rabbit fluorophore-conjugated secondary antibody.

    Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Incubation, Release Assay

    S309-CAR-NK-92MI cells bind to RBD domain of SARS-CoV-2 S protein and pseudotyped SARS-CoV-2 viral particles. ( a ) Representative dot plots showing the efficiency of S309-CAR binding to SARS-CoV-2-RBD. S309-CAR-NK-92MI or parental NK-92MI cells were incubated with the RBD recombinant protein of SARS-CoV-1 or SARS-CoV-2. ( b ) Generation of pseudotyped SARS-CoV-2 viral particles. 293T cells were transfected with various plasmids for 72 hoursfor the generation of pseudotyped SARS-CoV-2 viral particles. ( c ) Representative histogram showing S309-CAR-NK-92MI binds to the pseudotyped SARS-CoV-2 viral particles. S309-CAR-NK-92MI, parental NK-92MI, or 293T-hACE2 (positive control) cells were incubated with pseudotypedSARS-CoV-2 viral particles, S1 subunit, or full-length S recombinant protein at 37°C for 1 hour. The experimental sample was performed in triplicates with MFI = 13579 ± 251 (a.u.). ( d ) Quantitative data of the binding efficiency of S309-CAR-NK-92MI cells to pseudotyped SARS-CoV-2 viral particles. The experimental sample was performed in triplicates with binding efficiency of over 90%. Data represent mean ± standard error of the mean (SEM) of three independent experiments. Unpaired Student’s t test was employed. ****p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: S309-CAR-NK-92MI cells bind to RBD domain of SARS-CoV-2 S protein and pseudotyped SARS-CoV-2 viral particles. ( a ) Representative dot plots showing the efficiency of S309-CAR binding to SARS-CoV-2-RBD. S309-CAR-NK-92MI or parental NK-92MI cells were incubated with the RBD recombinant protein of SARS-CoV-1 or SARS-CoV-2. ( b ) Generation of pseudotyped SARS-CoV-2 viral particles. 293T cells were transfected with various plasmids for 72 hoursfor the generation of pseudotyped SARS-CoV-2 viral particles. ( c ) Representative histogram showing S309-CAR-NK-92MI binds to the pseudotyped SARS-CoV-2 viral particles. S309-CAR-NK-92MI, parental NK-92MI, or 293T-hACE2 (positive control) cells were incubated with pseudotypedSARS-CoV-2 viral particles, S1 subunit, or full-length S recombinant protein at 37°C for 1 hour. The experimental sample was performed in triplicates with MFI = 13579 ± 251 (a.u.). ( d ) Quantitative data of the binding efficiency of S309-CAR-NK-92MI cells to pseudotyped SARS-CoV-2 viral particles. The experimental sample was performed in triplicates with binding efficiency of over 90%. Data represent mean ± standard error of the mean (SEM) of three independent experiments. Unpaired Student’s t test was employed. ****p

    Article Snippet: The presence of the SARS-CoV-2 pseudovirus was further confirmed by flow cytometry, transfected 293T-hACE2 cells were stained with primary anti-RBD followed by goat anti-rabbit fluorophore-conjugated secondary antibody.

    Techniques: Binding Assay, Incubation, Recombinant, Transfection, Positive Control

    In vivo effects of cross-reactive antibodies ( A ) Timeline of the prophylactic antibody experiment in SARS-CoV-2 mouse adapted (MA) in vivo infection model. 200 μg antibody was given via intraperitoneal route to 12-month old female BALB/c mice 12 hours prior to virus inoculation (n= 4 or 5 per group). 1×10 3 or 1×10 4 PFU infectious dose of SARS-CoV-2 MA was administered intranasally for the low dose and high dose experiments, respectively. Weights were measured daily, and on day 4 tissue was collected for histopathology and viral load quantification. ( B ) Lung hemorrhage scores of gross pathology are shown for each low dose (1×10 3 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed. ( C ) For the experiment treating with 1×10 4 PFU of SARS-CoV-2 MA, percent survival for each antibody group is shown. 2/5, 4/5, 3/5, and 2/5 mice survived to day 4 for antibodies 46472-4, 46472-12, CR3022 and isotype control DENV-2D22 respectively. (D) Lung hemorrhage scores of gross pathology are shown for each high dose (1×10 4 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed.

    Journal: bioRxiv

    Article Title: Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

    doi: 10.1101/2020.12.20.414748

    Figure Lengend Snippet: In vivo effects of cross-reactive antibodies ( A ) Timeline of the prophylactic antibody experiment in SARS-CoV-2 mouse adapted (MA) in vivo infection model. 200 μg antibody was given via intraperitoneal route to 12-month old female BALB/c mice 12 hours prior to virus inoculation (n= 4 or 5 per group). 1×10 3 or 1×10 4 PFU infectious dose of SARS-CoV-2 MA was administered intranasally for the low dose and high dose experiments, respectively. Weights were measured daily, and on day 4 tissue was collected for histopathology and viral load quantification. ( B ) Lung hemorrhage scores of gross pathology are shown for each low dose (1×10 3 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed. ( C ) For the experiment treating with 1×10 4 PFU of SARS-CoV-2 MA, percent survival for each antibody group is shown. 2/5, 4/5, 3/5, and 2/5 mice survived to day 4 for antibodies 46472-4, 46472-12, CR3022 and isotype control DENV-2D22 respectively. (D) Lung hemorrhage scores of gross pathology are shown for each high dose (1×10 4 PFU of SARS-CoV-2 MA) treatment group. An ordinary one-way ANOVA test with multiple comparisons was performed.

    Article Snippet: For binding studies, SARS-CoV-2 HexaPro S, SARS-CoV-1 S, SARS-CoV-2 RBD, SARS-CoV-1 RBD, and MERS-CoV RBD constructs were expressed in the transient expression system previously mentioned.

    Techniques: In Vivo, Infection, Mouse Assay, Histopathology

    Functional activity of cross-reactive coronavirus antibodies ( A ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular phagocytosis activity (ADCP) against SARS-CoV-2 S, compared to positive control antibody CR3022 and negative control Palivizumab, an anti-RSV antibody. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3C . ( B ) 46472-4 and 46472-12 were tested for antibody-dependent cellular phagocytosis activity against SARS-CoV-1 S, compared to CR3022 antibody and anti-RSV antibody Palivizumab. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3D . ( C ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis (ADCT) activity against SARS-CoV-2 S coated on cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3E . ( D ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis activity against SARS-CoV-2 S displayed on transfected cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3F . ( E ) Cross-reactive coronavirus antibodies were tested for antibody-dependent complement deposition (ADCD) activity against SARS-CoV-2 S, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the C3b deposition score is shown, calculated from data in Figure S3G .

    Journal: bioRxiv

    Article Title: Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

    doi: 10.1101/2020.12.20.414748

    Figure Lengend Snippet: Functional activity of cross-reactive coronavirus antibodies ( A ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular phagocytosis activity (ADCP) against SARS-CoV-2 S, compared to positive control antibody CR3022 and negative control Palivizumab, an anti-RSV antibody. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3C . ( B ) 46472-4 and 46472-12 were tested for antibody-dependent cellular phagocytosis activity against SARS-CoV-1 S, compared to CR3022 antibody and anti-RSV antibody Palivizumab. Area under the curve of the phagocytosis score is shown, calculated from data in Figure S3D . ( C ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis (ADCT) activity against SARS-CoV-2 S coated on cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3E . ( D ) Cross-reactive coronavirus antibodies were tested for antibody-dependent cellular trogocytosis activity against SARS-CoV-2 S displayed on transfected cells, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the trogocytosis score is shown, calculated from data in Figure S3F . ( E ) Cross-reactive coronavirus antibodies were tested for antibody-dependent complement deposition (ADCD) activity against SARS-CoV-2 S, compared to positive control CR3022 and anti-RSV antibody Palivizumab. Area under the curve of the C3b deposition score is shown, calculated from data in Figure S3G .

    Article Snippet: For binding studies, SARS-CoV-2 HexaPro S, SARS-CoV-1 S, SARS-CoV-2 RBD, SARS-CoV-1 RBD, and MERS-CoV RBD constructs were expressed in the transient expression system previously mentioned.

    Techniques: Functional Assay, Activity Assay, Positive Control, Negative Control, Transfection

    Identification of coronavirus cross-reactive antibodies from SARS-CoV-1 convalescent PBMC sample using LIBRA-seq ( A ) Schematic of DNA-barcoded antigens used to probe a SARS-CoV-1 donor PBMC sample. The LIBRA-seq experiment setup consisted of eight oligo-labelled antigens in the screening library: SARS-CoV-2 S, SARS-CoV-1 S, MERS-CoV S, MERS-CoV S1, OC43-CoV S, HKU1-CoV S, and two HIV negative controls (ZM197, and CZA97). ( B ) LIBRA-seq scores for SARS-CoV-1 (x-axis) and SARS-CoV-2 (y-axis) for all IgG cells recovered from sequencing are shown as circles. The 15 lead antibody candidates are highlighted in purple. ( C ) Antibodies were tested for binding to SARS-CoV-2 S (S-2P), SARS-CoV-1 S (S-2P), OC43-CoV S (S-2P), HKU1-CoV S (S-2P), and SARS-CoV-2 S (HexaPro) by ELISA. HIV-specific antibody VRC01 is used as a negative control. Anti-SARS-CoV-1 mouse antibody 240CD was also used (BEI Resources). ELISAs were performed in technical duplicates with at least two biological duplicates. ( D ) ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure 1C , with AUC of 0 displayed as white, and maximum AUC as purple. ELISAs were performed in technical duplicates with at least two biological duplicates.

    Journal: bioRxiv

    Article Title: Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

    doi: 10.1101/2020.12.20.414748

    Figure Lengend Snippet: Identification of coronavirus cross-reactive antibodies from SARS-CoV-1 convalescent PBMC sample using LIBRA-seq ( A ) Schematic of DNA-barcoded antigens used to probe a SARS-CoV-1 donor PBMC sample. The LIBRA-seq experiment setup consisted of eight oligo-labelled antigens in the screening library: SARS-CoV-2 S, SARS-CoV-1 S, MERS-CoV S, MERS-CoV S1, OC43-CoV S, HKU1-CoV S, and two HIV negative controls (ZM197, and CZA97). ( B ) LIBRA-seq scores for SARS-CoV-1 (x-axis) and SARS-CoV-2 (y-axis) for all IgG cells recovered from sequencing are shown as circles. The 15 lead antibody candidates are highlighted in purple. ( C ) Antibodies were tested for binding to SARS-CoV-2 S (S-2P), SARS-CoV-1 S (S-2P), OC43-CoV S (S-2P), HKU1-CoV S (S-2P), and SARS-CoV-2 S (HexaPro) by ELISA. HIV-specific antibody VRC01 is used as a negative control. Anti-SARS-CoV-1 mouse antibody 240CD was also used (BEI Resources). ELISAs were performed in technical duplicates with at least two biological duplicates. ( D ) ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure 1C , with AUC of 0 displayed as white, and maximum AUC as purple. ELISAs were performed in technical duplicates with at least two biological duplicates.

    Article Snippet: For binding studies, SARS-CoV-2 HexaPro S, SARS-CoV-1 S, SARS-CoV-2 RBD, SARS-CoV-1 RBD, and MERS-CoV RBD constructs were expressed in the transient expression system previously mentioned.

    Techniques: Sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    Epitope mapping of cross-reactive antibodies ( A ) For cross-reactive coronavirus antibodies, ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2A and ( B ) for SARS-CoV-2 S1 reactive antibodies, ELISA binding data against the RBD and NTD are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2B . ELISA AUC is displayed as a heat map. AUC of 0 is displayed as white and maximum AUC as purple. ELISA data are representative of at least two independent experiments. Anti-HIV antibody VRC01 and anti-VEGF antibody are shown as a negative control and anti-SARS-CoV-1 antibody 240CD is shown as positive control. ( C ) Surface plasmon resonance binding of 46472-12 Fab to SARS-CoV-2 RBD. Affinity measurements are shown to the right of the graph. ( D ) Cross-reactive antibodies were used in a competition ELISA to determine if binding of one antibody affected binding of another. Competitor antibodies were added at 10 μg/ml, and then detected antibodies were added at 0.1 μg/ml. The percent reduction in binding compared to binding without a competitor is shown. An anti-HIV antibody was also used as a negative control. ELISAs were performed in technical duplicates with at least two biological duplicates. ( E ) Antibodies were tested for autoreactivity against a variety of antigens in the Luminex AtheNA assay. Anti-HIV antibody 4E10 was used as a positive control and Ab82 was used as a negative control. ( F ) Cross-reactive coronavirus antibodies target a variety of epitopes on the SARS-CoV-2 S protein, including the RBD, NTD, and S2 domains, highlighted on the structure (PDB: 6VSB). Antibodies targeting each epitope are listed and color coded for each domain.

    Journal: bioRxiv

    Article Title: Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions

    doi: 10.1101/2020.12.20.414748

    Figure Lengend Snippet: Epitope mapping of cross-reactive antibodies ( A ) For cross-reactive coronavirus antibodies, ELISA binding data against the antigens are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2A and ( B ) for SARS-CoV-2 S1 reactive antibodies, ELISA binding data against the RBD and NTD are displayed as a heatmap of the AUC analysis calculated from the data in Figure S2B . ELISA AUC is displayed as a heat map. AUC of 0 is displayed as white and maximum AUC as purple. ELISA data are representative of at least two independent experiments. Anti-HIV antibody VRC01 and anti-VEGF antibody are shown as a negative control and anti-SARS-CoV-1 antibody 240CD is shown as positive control. ( C ) Surface plasmon resonance binding of 46472-12 Fab to SARS-CoV-2 RBD. Affinity measurements are shown to the right of the graph. ( D ) Cross-reactive antibodies were used in a competition ELISA to determine if binding of one antibody affected binding of another. Competitor antibodies were added at 10 μg/ml, and then detected antibodies were added at 0.1 μg/ml. The percent reduction in binding compared to binding without a competitor is shown. An anti-HIV antibody was also used as a negative control. ELISAs were performed in technical duplicates with at least two biological duplicates. ( E ) Antibodies were tested for autoreactivity against a variety of antigens in the Luminex AtheNA assay. Anti-HIV antibody 4E10 was used as a positive control and Ab82 was used as a negative control. ( F ) Cross-reactive coronavirus antibodies target a variety of epitopes on the SARS-CoV-2 S protein, including the RBD, NTD, and S2 domains, highlighted on the structure (PDB: 6VSB). Antibodies targeting each epitope are listed and color coded for each domain.

    Article Snippet: For binding studies, SARS-CoV-2 HexaPro S, SARS-CoV-1 S, SARS-CoV-2 RBD, SARS-CoV-1 RBD, and MERS-CoV RBD constructs were expressed in the transient expression system previously mentioned.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Positive Control, SPR Assay, Luminex

    The proposed mechanism of ACE2-induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the closed ground prefusion state (with packed FP, in red) to the transiently open state (step 1) with an untwisting motion (highlighted in dark gray arrow) associated with a downward movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (step 2), and then all the way to the refolded postfusion state (step 3). The continuous swing motions of ACE2-RBD within S trimer are indicated by red arrows. The S trimer associated with ACE2 dimer (third panel) was generated by aligning the ACE2 of our S-ACE2 structure with the available full-length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a cartoon (PDB: 6XRA).

    Journal: Science Advances

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    doi: 10.1126/sciadv.abe5575

    Figure Lengend Snippet: The proposed mechanism of ACE2-induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the closed ground prefusion state (with packed FP, in red) to the transiently open state (step 1) with an untwisting motion (highlighted in dark gray arrow) associated with a downward movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (step 2), and then all the way to the refolded postfusion state (step 3). The continuous swing motions of ACE2-RBD within S trimer are indicated by red arrows. The S trimer associated with ACE2 dimer (third panel) was generated by aligning the ACE2 of our S-ACE2 structure with the available full-length ACE2 dimer structure (PDB: 6M1D). The postfusion state was illustrated as a cartoon (PDB: 6XRA).

    Article Snippet: For mutants RBD-(RBM-R2) and RBD-(RBM-R3), residues L452 to K462, and residues T470 to T478 of the RBM region in the SARS-CoV-2 RBD were mutated into the corresponding regions of SARS-CoV strain Tor2, respectively.

    Techniques: Generated

    A tightly closed conformation of SARS-CoV-2 S trimer. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. ( C ) Close-up view of the model map fitting in the NTD and RBD regions of the S1 subunit, illustrating that most of the NTD region was well resolved. ( D ) Overlaid RBD structures of our S-closed (blue) with a cryo-EM structure of SARS-CoV-2 S in closed state (6VXX, gray), illustrating that the RBM S469-C488 loop was captured in our structure (indicated by dotted ellipsoid). ( E ) Top view of the overlaid structures as in (D) (left) and zoom-in views of specific domains, showing that there is a marked counterclockwise rotation in S1 especially in NTD, resulting in a twisted, tightly closed conformation. ( F ) Protomer interaction interface analysis by PISA. ( G ) Location of the captured FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunits are colored steel blue and gold, respectively. ( H ) Model map fitting for the FP fragment. ( I ) Close-up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.

    Journal: Science Advances

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    doi: 10.1126/sciadv.abe5575

    Figure Lengend Snippet: A tightly closed conformation of SARS-CoV-2 S trimer. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S trimer in a tightly closed state, with three protomers shown in different color. ( C ) Close-up view of the model map fitting in the NTD and RBD regions of the S1 subunit, illustrating that most of the NTD region was well resolved. ( D ) Overlaid RBD structures of our S-closed (blue) with a cryo-EM structure of SARS-CoV-2 S in closed state (6VXX, gray), illustrating that the RBM S469-C488 loop was captured in our structure (indicated by dotted ellipsoid). ( E ) Top view of the overlaid structures as in (D) (left) and zoom-in views of specific domains, showing that there is a marked counterclockwise rotation in S1 especially in NTD, resulting in a twisted, tightly closed conformation. ( F ) Protomer interaction interface analysis by PISA. ( G ) Location of the captured FP fragment (in deep pink) within the S trimer (left) and one protomer. S1 and S2 subunits are colored steel blue and gold, respectively. ( H ) Model map fitting for the FP fragment. ( I ) Close-up view of the interactions between D614 from SD2 and FP, with the hydrogen bonds labeled in dotted lines and the L828-F855 region in FP in deep pink.

    Article Snippet: For mutants RBD-(RBM-R2) and RBD-(RBM-R3), residues L452 to K462, and residues T470 to T478 of the RBM region in the SARS-CoV-2 RBD were mutated into the corresponding regions of SARS-CoV strain Tor2, respectively.

    Techniques: Labeling

    The architecture of the SARS-CoV-2 S-ACE2 complex. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. ( C ) Side and top views of the overlaid S-open (color) and S-closed (dark gray) structures, showing that in the open process, there is a 71.0° upward/outward rotation of RBD associated with a downward shift of SD1 in protomer 1. ( D ) Rotations of NTD and CH from the S-closed (gray) to the S-open (in color) state, with the NTD also showing a downward/outward movement (right). ( E ) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing that the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2. ( F ) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. ( G ) Protomer interaction interface analysis of S-ACE2 by PISA. ( H ) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). ( I ) Overlaid structures of S-ACE2 (gray) and S-closed (color, with the FP fragment in deep pink), indicating a downward shift of SD1 and most of the FP is missing in S-ACE2. Close-up view (right) of the potential clashes between the downward-shifted SD1 β34 and α8 helix of FP. ( J ) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.

    Journal: Science Advances

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    doi: 10.1126/sciadv.abe5575

    Figure Lengend Snippet: The architecture of the SARS-CoV-2 S-ACE2 complex. ( A and B ) Cryo-EM map and model of SARS-CoV-2 S-ACE2 complex. We named the RBD up protomer as protomer 1 (light green), and the other two RBD down ones as protomer 2 (royal blue) and protomer 3 (gold). ACE2 was colored in violet red. ( C ) Side and top views of the overlaid S-open (color) and S-closed (dark gray) structures, showing that in the open process, there is a 71.0° upward/outward rotation of RBD associated with a downward shift of SD1 in protomer 1. ( D ) Rotations of NTD and CH from the S-closed (gray) to the S-open (in color) state, with the NTD also showing a downward/outward movement (right). ( E ) Side view of the overlaid S-ACE2 (violet red) and S-open (light green) protomer 1 structures, showing that the angle between the long axis of RBD and the horizontal plane of S trimer reduces from the S-open to the S-ACE2. ( F ) Top and side views of the overlaid S-ACE2 (violet red) and S-open (color) RBD structures, showing the coordinated movements of RBDs. ( G ) Protomer interaction interface analysis of S-ACE2 by PISA. ( H ) Aromatic interactions between the core region of the up RBD-1 (green) and the RBM T470-F490 loop of the neighboring RBD-2 (blue). ( I ) Overlaid structures of S-ACE2 (gray) and S-closed (color, with the FP fragment in deep pink), indicating a downward shift of SD1 and most of the FP is missing in S-ACE2. Close-up view (right) of the potential clashes between the downward-shifted SD1 β34 and α8 helix of FP. ( J ) Population shift between the ACE2-unpresented and ACE2-presented S trimer samples.

    Article Snippet: For mutants RBD-(RBM-R2) and RBD-(RBM-R3), residues L452 to K462, and residues T470 to T478 of the RBM region in the SARS-CoV-2 RBD were mutated into the corresponding regions of SARS-CoV strain Tor2, respectively.

    Techniques:

    The T470-T478 loop and residue Y505 within RBM play important roles in the engagement of SARS-CoV-2 spike with receptor ACE2. ( A ) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom-in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. ( B ) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting that the RBM T470-F490 loop has obvious conformational variations. ( C ) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left. Anti-RBD sera and a cross-reactive monoclonal antibody (MAb) 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared with wild-type RBD. ( D ) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein. OD 450 , optical density at 450 nm.

    Journal: Science Advances

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    doi: 10.1126/sciadv.abe5575

    Figure Lengend Snippet: The T470-T478 loop and residue Y505 within RBM play important roles in the engagement of SARS-CoV-2 spike with receptor ACE2. ( A ) The overall view of ACE2 (violet red) bound protomer 1 (light green) from our S-ACE2 structure, and zoom-in view of the interaction interface between ACE2 and RBD, with the key contacting elements T470-F490 loop and Q498-Y505 within RBM highlighted in black ellipsoid and blue ellipsoid, respectively. ( B ) Superposition of our SARS-CoV-2 S-ACE2 structure with the crystal structure of SARS-CoV RBD-ACE2 (PDB: 2AJF), suggesting that the RBM T470-F490 loop has obvious conformational variations. ( C ) Binding activities of ACE2-hFc fusion protein to wild-type (wt) and mutant SARS-CoV-2 RBD proteins determined by ELISA. Different structural elements of RBD were colored in the left. Anti-RBD sera and a cross-reactive monoclonal antibody (MAb) 1A10 served as positive controls. Ctr, an irrelevant antibody. The black arrow indicates that mutations in the RBD (RBM-R3) mutant significantly reduced the binding of ACE2-hFc compared with wild-type RBD. ( D ) Binding of ACE2-hFc fusion protein to wt and single-point mutant forms of SARS-CoV-2 RBD protein measured by ELISA. RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were mutated to Ala, respectively. The downward arrow indicates that the mutation at Y505 completely abolished the binding of ACE2 to RBD protein. OD 450 , optical density at 450 nm.

    Article Snippet: For mutants RBD-(RBM-R2) and RBD-(RBM-R3), residues L452 to K462, and residues T470 to T478 of the RBM region in the SARS-CoV-2 RBD were mutated into the corresponding regions of SARS-CoV strain Tor2, respectively.

    Techniques: Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay

    Organization of the resolved N-linked glycans of SARS-CoV-2 S trimer. ( A ) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. A total of 18 N-linked glycans detected in our S-closed cryo-EM map are shown in red, and the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). ( B ) Surface representation of the glycosylated S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black dotted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at the N17 and N149 sites are indicated in the top view. ( C ) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red.

    Journal: Science Advances

    Article Title: Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

    doi: 10.1126/sciadv.abe5575

    Figure Lengend Snippet: Organization of the resolved N-linked glycans of SARS-CoV-2 S trimer. ( A ) Schematic representation of SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons are shown as branches. A total of 18 N-linked glycans detected in our S-closed cryo-EM map are shown in red, and the remaining undetected ones in black. After ACE2 binding, the glycan density that appears weaker is indicated (*). ( B ) Surface representation of the glycosylated S trimer in the S-closed state with N-linked glycans shown in red. The location of glycan hole is indicated in black dotted ellipsoid, with the locations of S1/S2 and FP, and glycan at N657 site near the glycan hole indicated. The newly captured glycans at the N17 and N149 sites are indicated in the top view. ( C ) Surface representation of the glycosylated S-ACE2 complex with N-linked glycans in red.

    Article Snippet: For mutants RBD-(RBM-R2) and RBD-(RBM-R3), residues L452 to K462, and residues T470 to T478 of the RBM region in the SARS-CoV-2 RBD were mutated into the corresponding regions of SARS-CoV strain Tor2, respectively.

    Techniques: Binding Assay