sars cov2 s1  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike S1 His Recombinant Protein HPLC verified COVID 19 Spike S1 Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV spike protein S1 Subunit YP 009724390 1 Val16 Arg685 was expressed with a polyhistidine tag at the C terminus
    Catalog Number:
    40591-V08H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV spike protein S1 Subunit YP 009724390 1 Val16 Arg685 was expressed with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/sars cov2 s1/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov2 s1 - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors"

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

    Journal: Small Methods

    doi: 10.1002/smtd.202001031

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

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

    Related Articles

    Mouse Assay:

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors
    Article Snippet: .. Generation and Production of Antibodies against SARS‐CoV‐2 S Balb/c mice were intraperitoneal immunized with 5 µg of SARS‐CoV2‐RBD (expression in this study, n = 5), SARS‐CoV2‐S1 (Sino Biological, 40591‐V08H, n = 3), and SARS‐CoV2‐S2 (Sino Biological, 40590‐V08B, n = 3), respectively. ..

    Expressing:

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors
    Article Snippet: .. Generation and Production of Antibodies against SARS‐CoV‐2 S Balb/c mice were intraperitoneal immunized with 5 µg of SARS‐CoV2‐RBD (expression in this study, n = 5), SARS‐CoV2‐S1 (Sino Biological, 40591‐V08H, n = 3), and SARS‐CoV2‐S2 (Sino Biological, 40590‐V08B, n = 3), respectively. ..

    Multiplex Assay:

    Article Title: SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence
    Article Snippet: Assay Procedure The steps in assay validation were similar to recently developed bead-based multiplex immunoassays for CMV, EBV, and RSV, with minor modifications as described below [ , ]. .. For the multiplex bead-based immune assay the following antigens obtained from Sino Biological were used: SARS-CoV-2 monomeric spike S1 (40591-V08H), RBD (40592-V08B), and nucleoprotein (N) (40588-V08B). ..

    Recombinant:

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates
    Article Snippet: Serum and BAL fluid collected at each time point were evaluated for binding titers. .. Ninety-six-well immunosorbent plates (NUNC) were coated with 1 μg/mL recombinant SARS-CoV-2 S1+S2 ECD protein (Sino Biological 40589-V08B1) and S1 protein (Sino Biological 40591-V08H) in PBS (phosphate-buffered saline) overnight at 4 °C. ..

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    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Sars Cov2 S1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    99
    Sino Biological anti s2
    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using <t>anti-S2</t> Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P
    Anti S2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Sino Biological sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research
    Adjuvanted S and S1 immune sera exhibit high titers of <t>SARS-CoV-2</t> pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p
    Sars Cov 2 2019 Ncov Spike S1 S2 Ecd His Recombinant Protein Covid 19 Spike Research, 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 s1 his proteins from sars cov 2
    Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on <t>S1-His</t> with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.
    S1 His Proteins From Sars Cov 2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

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

    doi: 10.1002/smtd.202001031

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

    Article Snippet: Generation and Production of Antibodies against SARS‐CoV‐2 S Balb/c mice were intraperitoneal immunized with 5 µg of SARS‐CoV2‐RBD (expression in this study, n = 5), SARS‐CoV2‐S1 (Sino Biological, 40591‐V08H, n = 3), and SARS‐CoV2‐S2 (Sino Biological, 40590‐V08B, n = 3), respectively.

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

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

    Journal: bioRxiv

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

    doi: 10.1101/2020.10.15.339838

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

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

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

    Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Neutralization, Binding Assay, Inhibition, Luciferase, Activity Assay, Infection, Expressing, Mouse Assay, Irradiation, Incubation

    B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Mouse Assay, Enzyme-linked Immunospot, In Vitro, Flow Cytometry, Staining

    SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Binding Assay, Concentration Assay

    Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.

    Journal: Nature Communications

    Article Title: SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

    doi: 10.1038/s41467-021-21609-2

    Figure Lengend Snippet: Binding to RBD mutants, epitopes, and structure models. A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.

    Article Snippet: S1-HIS proteins from SARS-CoV-2 (expressed in HEK cells), SARS-CoV-1, MERS, HCoV HKU1, HCoV NL63, and HCoV 229E were acquired commercially (Sino Biologicals products 40591-V08H, 40150-V08B1, 40069-V08H, 40021-V08H, 40601-V08H, 40600-V08H).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Protein Array, Sequencing

    Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).

    Journal: Nature Communications

    Article Title: SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

    doi: 10.1038/s41467-021-21609-2

    Figure Lengend Snippet: Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).

    Article Snippet: S1-HIS proteins from SARS-CoV-2 (expressed in HEK cells), SARS-CoV-1, MERS, HCoV HKU1, HCoV NL63, and HCoV 229E were acquired commercially (Sino Biologicals products 40591-V08H, 40150-V08B1, 40069-V08H, 40021-V08H, 40601-V08H, 40600-V08H).

    Techniques: Neutralization, Titration, Enzyme-linked Immunosorbent Assay, Produced, SPR Assay