sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD His 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 a polyhistidine tag at the C terminus
    Catalog Number:
    40592-V08B
    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:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of <t>SARS-CoV-2</t> S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with a polyhistidine tag at the C terminus
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    1) Product Images from "Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies"

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    Journal: bioRxiv

    doi: 10.1101/2021.04.03.438258

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Figure Legend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Techniques Used: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.
    Figure Legend Snippet: SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Techniques Used: Neutralization, Stable Transfection, Expressing, Infection

    RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).
    Figure Legend Snippet: RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Techniques Used: Incubation, Staining, Imaging, Expressing, Fluorescence, Cryo-EM Sample Prep, Inhibition

    S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).
    Figure Legend Snippet: S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Techniques Used: In Vivo, Injection, Infection, TCID50 Assay

    SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD
    Figure Legend Snippet: SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Techniques Used: Activity Assay, Neutralization, Infection, Incubation, Stable Transfection, Immunostaining, Inhibition, Activation Assay, Reporter Assay

    Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.
    Figure Legend Snippet: Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Techniques Used: Expressing, Infection, In Vitro, Transformation Assay, Transduction, Incubation, Cell Culture, Adsorption, Blocking Assay

    Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.
    Figure Legend Snippet: Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.

    Techniques Used: Neutralization, Infection, Purification, Incubation, Protein Binding, Flow Cytometry

    Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p
    Figure Legend Snippet: Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Techniques Used: Injection, Infection

    HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.
    Figure Legend Snippet: HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Techniques Used: Expressing, Infection, Stable Transfection

    Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.
    Figure Legend Snippet: Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Techniques Used: Binding Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence, Infection, Variant Assay

    DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.
    Figure Legend Snippet: DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.

    Techniques Used: Infection, Transfection, Stable Transfection, Luciferase, Incubation

    Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.
    Figure Legend Snippet: Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Techniques Used:

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19
    Article Snippet: .. Enzyme-linked immunosorbent assay As previously described, 50 ng of SARS-CoV-2 S1 protein (Sino Biological, 40591-V08H) or SARS-CoV-2 RBD protein (Sino Biological, 40592-V08B) or SARS-CoV-2 S2 protein (Sino Biological, 40590-V08B) in 100 μl phosphate-buffered saline (PBS) per well was coated on ELISA plates (Costar, 42592) overnight at 4 °C. .. The ELISA plates were blocked for 1 h with 100 μl blocking buffer (5% fetal bovine serum (FBS) and 0.1% Tween 20 in PBS) and then incubated with diluted patient or healthy control sera in 100 μl blocking buffer for 1 h. After washing with PBST buffer (0.1% Tween 20 in PBS), the ELISA plates were incubated with anti-human IgG horseradish peroxidase (HRP) antibody (Bioss Biotech, 0297D) for 45 min, followed by PBST washing and addition of 3,3′,5,5′-tetramethylbenzidine (TMB) buffer (Beyotime, P0209).

    Flow Cytometry:

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: Cells were washed three times, resuspended in 200μL of FACS buffer and analyzed by flow cytometry using the CytoFLEX flow cytometer (Beckman Coulter). .. Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature. .. Cells were dissociated with TrpLE Express (Gibco, 12605-010) and 105 cells were transferred to each well of a 96-well V bottom plate (Corning, 3894).

    Binding Assay:

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: Cells were washed three times, resuspended in 200μL of FACS buffer and analyzed by flow cytometry using the CytoFLEX flow cytometer (Beckman Coulter). .. Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature. .. Cells were dissociated with TrpLE Express (Gibco, 12605-010) and 105 cells were transferred to each well of a 96-well V bottom plate (Corning, 3894).

    Generated:

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: Cells were washed three times, resuspended in 200μL of FACS buffer and analyzed by flow cytometry using the CytoFLEX flow cytometer (Beckman Coulter). .. Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature. .. Cells were dissociated with TrpLE Express (Gibco, 12605-010) and 105 cells were transferred to each well of a 96-well V bottom plate (Corning, 3894).

    Incubation:

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: Cells were washed three times, resuspended in 200μL of FACS buffer and analyzed by flow cytometry using the CytoFLEX flow cytometer (Beckman Coulter). .. Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature. .. Cells were dissociated with TrpLE Express (Gibco, 12605-010) and 105 cells were transferred to each well of a 96-well V bottom plate (Corning, 3894).

    other:

    Article Title: The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection
    Article Snippet: Reagents SARS-CoV-2 (2019-nCoV) Spike Protein (RBD, His Tag) (Sino Biologicals, 40592-V08B), and proteins were biotinylated using EZ-Link™ Sulfo-NHS-Biotin, No-Weigh™ (Thermo Fisher Scientific, A39256).

    Article Title: The Characterization of Disease Severity Associated IgG Subclasses Response in COVID-19 Patients
    Article Snippet: ProteinsSARS-CoV-2 Spike Protein (S1+S2) (cat# 40589-VO8B1), SARS-CoV-2 Spike RBD Protein (cat# 40592-V08B), SARS-CoV-2 Nucleocapsid Protein (cat# 40588-V08B) were purchased from Sino Biological (China).

    Microscale Thermophoresis:

    Article Title: Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs
    Article Snippet: The reaction was developed by adding 100 μL of tetramethylbenzidine (Neogen, Lansing, MI, USA) for 5 min at RT and measured at 450 nm by the microplate reader Victor Nivo (Perkin Elmer, Woodbridge, ON, Canada). .. Microscale Thermophoresis (MST)MST experiments were performed on a Monolith NT 115 system (Nano Temper Technologies, Munchen, Germany) and designed to evaluate the ability of the PPE to bind ACE2, S protein, and RBD (10108-H08H, 40589-V08B, 40592-V08B from Sino Biological, Wayne, PA, USA). .. The proteins used in the study were: ACE2 (NP_068576.1) (Met1-Ser740), Spike FL (YP_009724390.1) (Val16-Pro1296), and RBD Spike (YP_009724390.1) (Arg319-Phe541); all three produced as recombinant in baculovirus-insect cells and carrying a polyhistidine tag at the C-terminus.

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    Sino Biological recombinant biotinylated sars cov 2 spike protein rbd
    dACE2 is induced by <t>SARS-CoV-2</t> in human cell lines and organoid cultures. Expression of ACE2 , dACE2 and a control ISG IFIT1 in A) colon cancer cell lines Caco2 and T84 and a lung cancer cell line Calu3 and C) colon and ileum organoid cultures derived from two donors; B and D) SARS-CoV-2 viral loads in corresponding cells. P-values are for Student’s T-test.
    Recombinant Biotinylated Sars Cov 2 Spike Protein Rbd, 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
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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of <t>SARS-CoV-2</t> S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Sars Cov 2 2019 Ncov Spike Rbd His Recombinant Protein Covid 19 Spike Rbd Research, 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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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein biotinylated covid 19 spike rbd research
    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of <t>SARS-CoV-2</t> S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Sars Cov 2 2019 Ncov Spike Rbd His Recombinant Protein Biotinylated Covid 19 Spike Rbd Research, 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
    https://www.bioz.com/result/sars cov 2 2019 ncov spike rbd his recombinant protein biotinylated covid 19 spike rbd research/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov spike rbd his recombinant protein biotinylated covid 19 spike rbd research - by Bioz Stars, 2021-06
    94/100 stars
      Buy from Supplier

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    dACE2 is induced by SARS-CoV-2 in human cell lines and organoid cultures. Expression of ACE2 , dACE2 and a control ISG IFIT1 in A) colon cancer cell lines Caco2 and T84 and a lung cancer cell line Calu3 and C) colon and ileum organoid cultures derived from two donors; B and D) SARS-CoV-2 viral loads in corresponding cells. P-values are for Student’s T-test.

    Journal: bioRxiv

    Article Title: Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

    doi: 10.1101/2020.07.19.210955

    Figure Lengend Snippet: dACE2 is induced by SARS-CoV-2 in human cell lines and organoid cultures. Expression of ACE2 , dACE2 and a control ISG IFIT1 in A) colon cancer cell lines Caco2 and T84 and a lung cancer cell line Calu3 and C) colon and ileum organoid cultures derived from two donors; B and D) SARS-CoV-2 viral loads in corresponding cells. P-values are for Student’s T-test.

    Article Snippet: After 24 hrs, cells were treated with 2 ng/ml of recombinant biotinylated SARS-CoV-2 spike protein RBD (spike protein-RBD, Sino Biological) for 1 hour at 37°C.

    Techniques: Expressing, Derivative Assay

    dACE2 is non-functional for binding SARS-CoV-2 spike protein RBD and as a carboxypeptidase. A) Representative confocal images of T24 cells transiently overexpressing dACE2-Myc (white), ACE2-GFP (green) and treated with receptor-binding domain (RBD) of SARS-CoV-2 spike protein (red), nuclei (DAPI)-blue; bars=20μM. B-D) Representative flow cytometry histogram B) and mean fluorescence intensity (MFI) values from 3 biological replicates C) of spike-RBD binding to the surface of ACE2-GFP but not dACE2-Myc expressing T24 cells. D) Representative flow cytometry plot showing gates for T24 cells co-transfected with dACE2-Myc and ACE2-GFP. Gates were drawn to identify cells expressing dACE2-Myc, ACE2-GFP, or both proteins. E) Histogram and F) plot depicting MFI of spike protein-RBD binding in gated cells from plot E , based on 3 biological replicates. Data represent one of two independent experiments. G) A representative Western blot showing detection of ACE2-GFP and dACE-GFP in protein lysates of T24 cells transfected with equal amounts of plasmids. Based on Western blot densitometry, equal estimated amounts of ACE2 and dACE2 proteins were used in peptidase activity assays in H . Results are based on 3 biological replicates.

    Journal: bioRxiv

    Article Title: Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

    doi: 10.1101/2020.07.19.210955

    Figure Lengend Snippet: dACE2 is non-functional for binding SARS-CoV-2 spike protein RBD and as a carboxypeptidase. A) Representative confocal images of T24 cells transiently overexpressing dACE2-Myc (white), ACE2-GFP (green) and treated with receptor-binding domain (RBD) of SARS-CoV-2 spike protein (red), nuclei (DAPI)-blue; bars=20μM. B-D) Representative flow cytometry histogram B) and mean fluorescence intensity (MFI) values from 3 biological replicates C) of spike-RBD binding to the surface of ACE2-GFP but not dACE2-Myc expressing T24 cells. D) Representative flow cytometry plot showing gates for T24 cells co-transfected with dACE2-Myc and ACE2-GFP. Gates were drawn to identify cells expressing dACE2-Myc, ACE2-GFP, or both proteins. E) Histogram and F) plot depicting MFI of spike protein-RBD binding in gated cells from plot E , based on 3 biological replicates. Data represent one of two independent experiments. G) A representative Western blot showing detection of ACE2-GFP and dACE-GFP in protein lysates of T24 cells transfected with equal amounts of plasmids. Based on Western blot densitometry, equal estimated amounts of ACE2 and dACE2 proteins were used in peptidase activity assays in H . Results are based on 3 biological replicates.

    Article Snippet: After 24 hrs, cells were treated with 2 ng/ml of recombinant biotinylated SARS-CoV-2 spike protein RBD (spike protein-RBD, Sino Biological) for 1 hour at 37°C.

    Techniques: Functional Assay, Binding Assay, Flow Cytometry, Fluorescence, Expressing, Transfection, Western Blot, Activity Assay

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Neutralization, Stable Transfection, Expressing, Infection

    RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Incubation, Staining, Imaging, Expressing, Fluorescence, Cryo-EM Sample Prep, Inhibition

    S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: In Vivo, Injection, Infection, TCID50 Assay

    SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Activity Assay, Neutralization, Infection, Incubation, Stable Transfection, Immunostaining, Inhibition, Activation Assay, Reporter Assay

    Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Expressing, Infection, In Vitro, Transformation Assay, Transduction, Incubation, Cell Culture, Adsorption, Blocking Assay

    Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Injection, Infection

    HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Expressing, Infection, Stable Transfection

    Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: Binding Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence, Infection, Variant Assay

    Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Article Snippet: Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells Biotinylated SARS-CoV-2 Spike D614G protein (Spikebiotin, in-house generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain (RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor® 647 streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for 20 min at room temperature.

    Techniques: