anti s2  (Sino Biological)


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    Sino Biological anti s2
    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using <t>anti-S2</t> Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p
    Anti S2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti s2/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti s2 - by Bioz Stars, 2021-05
    94/100 stars

    Images

    1) Product Images from "Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration"

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

    Journal: eLife

    doi: 10.7554/eLife.61552

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

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

    2) Product Images from "CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells"

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.14.426742

    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
    Figure Legend 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

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

    3) Product Images from "SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development"

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development

    Journal: bioRxiv

    doi: 10.1101/2020.02.16.951723

    Structural conservation of SARS-CoV RBD. RBD is shown as colored surface. ACE2 is shown as gray cartoon. The three surface mutation sites (i.e. N354D, D364Y, and V367F) observed in SARS-CoV-2 RBD are labeled. Mutation F342L is buried and not shown here.
    Figure Legend Snippet: Structural conservation of SARS-CoV RBD. RBD is shown as colored surface. ACE2 is shown as gray cartoon. The three surface mutation sites (i.e. N354D, D364Y, and V367F) observed in SARS-CoV-2 RBD are labeled. Mutation F342L is buried and not shown here.

    Techniques Used: Mutagenesis, Labeling

    Structure similarity between SARS-CoV-2 RBD and SARS-CoV RBD. RBD is shown in a space-filled model with colored surface. ACE2 is shown as gray tube model. The three glycosylation sites in SARS-CoV are labeled. Note that N 357 ST in SARS-CoV is changed to N 370 SA in SARS-CoV-2, which is different from the NXS/T pattern required for glycosylation, and hence this site is more likely to be unglycosylated. The two possible cross-reactive regions are marked with yellow circles.
    Figure Legend Snippet: Structure similarity between SARS-CoV-2 RBD and SARS-CoV RBD. RBD is shown in a space-filled model with colored surface. ACE2 is shown as gray tube model. The three glycosylation sites in SARS-CoV are labeled. Note that N 357 ST in SARS-CoV is changed to N 370 SA in SARS-CoV-2, which is different from the NXS/T pattern required for glycosylation, and hence this site is more likely to be unglycosylated. The two possible cross-reactive regions are marked with yellow circles.

    Techniques Used: Labeling

    Cross-reactivity and neutralization efficiency of SARS nAbs against SARS-CoV-2. A. Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. C. Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV. D. Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.
    Figure Legend Snippet: Cross-reactivity and neutralization efficiency of SARS nAbs against SARS-CoV-2. A. Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. C. Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV. D. Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.

    Techniques Used: Neutralization, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    Sequence analysis and structure modeling of SARS-CoV-2 RBD and SARS-CoV RBD and their interactions with ACE2. A. RBD sequence alignment of SARS-CoV and SARS-CoV-2, highlighting the predominant residues that contribute to the interactions with ACE2. The distinct interactions of RBD and ACE2 for the two viruses are indicated by the down-pointing orange triangles and up-pointing red triangles, respectively. RBM residues are underlined. The one-residue insertion is indicated by the red arrow. Asterisks indicate positions of fully conserved residues. Colons indicate positions of strictly conserved residues. Periods indicate positions of weakly conserved residues. B. Conformational comparison between the RBD-ACE2 complex structures for SARS-CoV-2 and SARS-CoV. The RBD and ACE2 structures in the SARS-CoV-2 RBD-ACE2 complex model are shown as orange and pink tubes, respectively. The RBD and ACE2 structures in the optimized SARS-CoV RBD-ACE2 complex structure are shown as blue and green tubes, respectively. The location of noticeable subtle conformational difference is indicated by an arrow. C. Distinct interaction patterns in the SARS-CoV-2 and SARS-CoV RBD-ACE2 interfaces. Structures of RBD and ACE2 are shown as cartoon in pink and green colors, respectively. The side chains of the residues in both protein components, representing their unique interactions, are shown as sticks. Polar interactions (salt-bridge and hydrogen bond) are shown as blue dash line. Non-polar interactions (π-stack, π-anion, and hydrophobic interactions) are shown as orange dash line.
    Figure Legend Snippet: Sequence analysis and structure modeling of SARS-CoV-2 RBD and SARS-CoV RBD and their interactions with ACE2. A. RBD sequence alignment of SARS-CoV and SARS-CoV-2, highlighting the predominant residues that contribute to the interactions with ACE2. The distinct interactions of RBD and ACE2 for the two viruses are indicated by the down-pointing orange triangles and up-pointing red triangles, respectively. RBM residues are underlined. The one-residue insertion is indicated by the red arrow. Asterisks indicate positions of fully conserved residues. Colons indicate positions of strictly conserved residues. Periods indicate positions of weakly conserved residues. B. Conformational comparison between the RBD-ACE2 complex structures for SARS-CoV-2 and SARS-CoV. The RBD and ACE2 structures in the SARS-CoV-2 RBD-ACE2 complex model are shown as orange and pink tubes, respectively. The RBD and ACE2 structures in the optimized SARS-CoV RBD-ACE2 complex structure are shown as blue and green tubes, respectively. The location of noticeable subtle conformational difference is indicated by an arrow. C. Distinct interaction patterns in the SARS-CoV-2 and SARS-CoV RBD-ACE2 interfaces. Structures of RBD and ACE2 are shown as cartoon in pink and green colors, respectively. The side chains of the residues in both protein components, representing their unique interactions, are shown as sticks. Polar interactions (salt-bridge and hydrogen bond) are shown as blue dash line. Non-polar interactions (π-stack, π-anion, and hydrophobic interactions) are shown as orange dash line.

    Techniques Used: Sequencing

    Measurements of SARS-CoV-2 and SARS-CoV S1 binding to ACE2. A. Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation. B. Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.
    Figure Legend Snippet: Measurements of SARS-CoV-2 and SARS-CoV S1 binding to ACE2. A. Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation. B. Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.

    Techniques Used: Binding Assay, Recombinant, Incubation, FACS

    Related Articles

    Generated:

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development
    Article Snippet: Hygromycin (Cat: V900372) were purchased from Sigma-Aldrich. .. SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV. .. Luciferase assay system (Cat: E1501) was purchased from Promega.

    Mouse Assay:

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development
    Article Snippet: Hygromycin (Cat: V900372) were purchased from Sigma-Aldrich. .. SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV. .. Luciferase assay system (Cat: E1501) was purchased from Promega.

    Recombinant:

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development
    Article Snippet: Hygromycin (Cat: V900372) were purchased from Sigma-Aldrich. .. SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV. .. Luciferase assay system (Cat: E1501) was purchased from Promega.

    Expressing:

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells
    Article Snippet: After 2 – 3 days, transduced cells were changed to fresh DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning). .. The spike protein expression was determined by flow cytometry by staining the transduced cells with anti-RBD antibody (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody. .. A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

    Flow Cytometry:

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells
    Article Snippet: After 2 – 3 days, transduced cells were changed to fresh DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning). .. The spike protein expression was determined by flow cytometry by staining the transduced cells with anti-RBD antibody (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody. .. A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

    Staining:

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells
    Article Snippet: After 2 – 3 days, transduced cells were changed to fresh DMEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL Penicillin-Streptomycin (Corning). .. The spike protein expression was determined by flow cytometry by staining the transduced cells with anti-RBD antibody (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody. .. A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

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    Sino Biological anti s2
    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. ( A ) Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] - and [N] - 293 T cells. All nine viruses were applied at equal titer to stable 293T/ACE2. ( B–C ) O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. ( D ) Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. ( E ) Western blot of Spike protein using <t>anti-S2</t> Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] - 293T products due to truncation of glycan biosynthesis. ( F ) Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] - 293Ts is almost fully proteolyzed during viral production (red arrowhead). *p
    Anti S2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti s2/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti s2 - by Bioz Stars, 2021-05
    94/100 stars
      Buy from Supplier

    86
    Sino Biological sars cov 2 2019 ncov spike neutralizing antibody mouse mab
    NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to <t>SARS-CoV-2</t> Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.
    Sars Cov 2 2019 Ncov Spike Neutralizing Antibody Mouse Mab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, 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 neutralizing antibody mouse mab/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov spike neutralizing antibody mouse mab - by Bioz Stars, 2021-05
    86/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: eLife

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

    doi: 10.7554/eLife.61552

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

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

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

    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 spike protein expression was determined by flow cytometry by staining the transduced cells with anti-RBD antibody (SinoBiological) followed by a goat anti-rabbit fluorophore-conjugated secondary antibody.

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

    Structural conservation of SARS-CoV RBD. RBD is shown as colored surface. ACE2 is shown as gray cartoon. The three surface mutation sites (i.e. N354D, D364Y, and V367F) observed in SARS-CoV-2 RBD are labeled. Mutation F342L is buried and not shown here.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development

    doi: 10.1101/2020.02.16.951723

    Figure Lengend Snippet: Structural conservation of SARS-CoV RBD. RBD is shown as colored surface. ACE2 is shown as gray cartoon. The three surface mutation sites (i.e. N354D, D364Y, and V367F) observed in SARS-CoV-2 RBD are labeled. Mutation F342L is buried and not shown here.

    Article Snippet: SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV.

    Techniques: Mutagenesis, Labeling

    Structure similarity between SARS-CoV-2 RBD and SARS-CoV RBD. RBD is shown in a space-filled model with colored surface. ACE2 is shown as gray tube model. The three glycosylation sites in SARS-CoV are labeled. Note that N 357 ST in SARS-CoV is changed to N 370 SA in SARS-CoV-2, which is different from the NXS/T pattern required for glycosylation, and hence this site is more likely to be unglycosylated. The two possible cross-reactive regions are marked with yellow circles.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development

    doi: 10.1101/2020.02.16.951723

    Figure Lengend Snippet: Structure similarity between SARS-CoV-2 RBD and SARS-CoV RBD. RBD is shown in a space-filled model with colored surface. ACE2 is shown as gray tube model. The three glycosylation sites in SARS-CoV are labeled. Note that N 357 ST in SARS-CoV is changed to N 370 SA in SARS-CoV-2, which is different from the NXS/T pattern required for glycosylation, and hence this site is more likely to be unglycosylated. The two possible cross-reactive regions are marked with yellow circles.

    Article Snippet: SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV.

    Techniques: Labeling

    Cross-reactivity and neutralization efficiency of SARS nAbs against SARS-CoV-2. A. Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. C. Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV. D. Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development

    doi: 10.1101/2020.02.16.951723

    Figure Lengend Snippet: Cross-reactivity and neutralization efficiency of SARS nAbs against SARS-CoV-2. A. Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein. C. Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV. D. Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.

    Article Snippet: SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV.

    Techniques: Neutralization, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    Sequence analysis and structure modeling of SARS-CoV-2 RBD and SARS-CoV RBD and their interactions with ACE2. A. RBD sequence alignment of SARS-CoV and SARS-CoV-2, highlighting the predominant residues that contribute to the interactions with ACE2. The distinct interactions of RBD and ACE2 for the two viruses are indicated by the down-pointing orange triangles and up-pointing red triangles, respectively. RBM residues are underlined. The one-residue insertion is indicated by the red arrow. Asterisks indicate positions of fully conserved residues. Colons indicate positions of strictly conserved residues. Periods indicate positions of weakly conserved residues. B. Conformational comparison between the RBD-ACE2 complex structures for SARS-CoV-2 and SARS-CoV. The RBD and ACE2 structures in the SARS-CoV-2 RBD-ACE2 complex model are shown as orange and pink tubes, respectively. The RBD and ACE2 structures in the optimized SARS-CoV RBD-ACE2 complex structure are shown as blue and green tubes, respectively. The location of noticeable subtle conformational difference is indicated by an arrow. C. Distinct interaction patterns in the SARS-CoV-2 and SARS-CoV RBD-ACE2 interfaces. Structures of RBD and ACE2 are shown as cartoon in pink and green colors, respectively. The side chains of the residues in both protein components, representing their unique interactions, are shown as sticks. Polar interactions (salt-bridge and hydrogen bond) are shown as blue dash line. Non-polar interactions (π-stack, π-anion, and hydrophobic interactions) are shown as orange dash line.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development

    doi: 10.1101/2020.02.16.951723

    Figure Lengend Snippet: Sequence analysis and structure modeling of SARS-CoV-2 RBD and SARS-CoV RBD and their interactions with ACE2. A. RBD sequence alignment of SARS-CoV and SARS-CoV-2, highlighting the predominant residues that contribute to the interactions with ACE2. The distinct interactions of RBD and ACE2 for the two viruses are indicated by the down-pointing orange triangles and up-pointing red triangles, respectively. RBM residues are underlined. The one-residue insertion is indicated by the red arrow. Asterisks indicate positions of fully conserved residues. Colons indicate positions of strictly conserved residues. Periods indicate positions of weakly conserved residues. B. Conformational comparison between the RBD-ACE2 complex structures for SARS-CoV-2 and SARS-CoV. The RBD and ACE2 structures in the SARS-CoV-2 RBD-ACE2 complex model are shown as orange and pink tubes, respectively. The RBD and ACE2 structures in the optimized SARS-CoV RBD-ACE2 complex structure are shown as blue and green tubes, respectively. The location of noticeable subtle conformational difference is indicated by an arrow. C. Distinct interaction patterns in the SARS-CoV-2 and SARS-CoV RBD-ACE2 interfaces. Structures of RBD and ACE2 are shown as cartoon in pink and green colors, respectively. The side chains of the residues in both protein components, representing their unique interactions, are shown as sticks. Polar interactions (salt-bridge and hydrogen bond) are shown as blue dash line. Non-polar interactions (π-stack, π-anion, and hydrophobic interactions) are shown as orange dash line.

    Article Snippet: SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV.

    Techniques: Sequencing

    Measurements of SARS-CoV-2 and SARS-CoV S1 binding to ACE2. A. Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation. B. Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development

    doi: 10.1101/2020.02.16.951723

    Figure Lengend Snippet: Measurements of SARS-CoV-2 and SARS-CoV S1 binding to ACE2. A. Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation. B. Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.

    Article Snippet: SARS-CoV neutralizing antibodies were generated from mice (M103, M127) or rabbits (R314, R301, R325, R302, R258, R348) immunized with recombinant S1 protein of SARS-CoV.

    Techniques: Binding Assay, Recombinant, Incubation, FACS

    NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to SARS-CoV-2 Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.

    Journal: ACS Nano

    Article Title: Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis

    doi: 10.1021/acsnano.0c05975

    Figure Lengend Snippet: NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to SARS-CoV-2 Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.

    Article Snippet: SARS-CoV S1-His (40150-V08B1), SARS-CoV-2 S1S2 ECD-His (40589-V08B1), SARS-CoV-2 S1-His (40591-V08H), SARS-CoV-2 RBD-His (40592-V08B), anti-SARS-CoV-2 S1 neutralizing antibody mouse mAb Ab1 (40591-MM43), and anti-SARS-CoV-2 RBD neutralizing antibody mouse mAb Ab2 (40592-MM57) were purchased from Sino Biological.

    Techniques: Inhibition, Blocking Assay