sars cov 2 2019 ncov nucleocapsid antibody rabbit pab  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Nucleocapsid Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant SARS CoV 2 2019 nCoV Nucleocapsid Protein Catalog 40588 V08B YP 009724397 2 335Gly Ala Met1 Ala419 The specific IgG was purified by SARS CoV 2 2019 nCoV Nucleocapsid affinity chromatography
    Catalog Number:
    40588-t62
    Product Aliases:
    Anti-coronavirus NP Antibody, Anti-coronavirus Nucleocapsid Antibody, Anti-coronavirus Nucleoprotein Antibody, Anti-cov np Antibody, Anti-ncov NP Antibody, Anti-NCP-CoV Nucleocapsid Antibody, Anti-novel coronavirus NP Antibody, Anti-novel coronavirus Nucleocapsid Antibody, Anti-novel coronavirus Nucleoprotein Antibody, Anti-np Antibody, Anti-nucleocapsid Antibody, Anti-Nucleoprotein Antibody
    Price:
    None
    Applications:
    WB,ELISA
    Host:
    Rabbit
    Immunogen:
    Recombinant SARS-CoV-2 / 2019-nCoV Nucleocapsid Protein (Catalog#40588-V08B)
    Category:
    Primary Antibody
    Antibody Type:
    PAb
    Isotype:
    Rabbit IgG
    Reactivity:
    2019 nCoV
    		Buy from Supplier

    Structured Review

    Sino Biological sars cov 2 2019 ncov nucleocapsid antibody rabbit pab
    ILRUN supresses <t>SARS-CoV-2</t> infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p
    Produced in rabbits immunized with purified recombinant SARS CoV 2 2019 nCoV Nucleocapsid Protein Catalog 40588 V08B YP 009724397 2 335Gly Ala Met1 Ala419 The specific IgG was purified by SARS CoV 2 2019 nCoV Nucleocapsid affinity chromatography
    https://www.bioz.com/result/sars cov 2 2019 ncov nucleocapsid antibody rabbit pab/product/Sino Biological
    Average 94 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov nucleocapsid antibody rabbit pab - by Bioz Stars, 2021-02
    94/100 stars

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    Images

    1) Product Images from "ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2"

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.11.13.381343

    ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p
    Figure Legend Snippet: ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p

    Techniques Used: Infection, Transfection

    Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p
    Figure Legend Snippet: Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p

    Techniques Used: Infection, Transfection

    2) Product Images from "Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery"

    Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

    Journal: bioRxiv

    doi: 10.1101/2020.08.02.233064

    Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.
    Figure Legend Snippet: Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.

    Techniques Used: Library Screening, Infection, Plasmid Preparation, Generated, Electric Cell-substrate Impedance Sensing, Concentration Assay, Staining

    SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors
    Figure Legend Snippet: SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors

    Techniques Used: Infection, Modification, Immunostaining, Plasmid Preparation, Generated

    3) Product Images from "Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery"

    Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

    Journal: bioRxiv

    doi: 10.1101/2020.08.02.233064

    Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.
    Figure Legend Snippet: Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.

    Techniques Used: Library Screening, Infection, Plasmid Preparation, Generated, Electric Cell-substrate Impedance Sensing, Concentration Assay, Staining

    SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors
    Figure Legend Snippet: SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors

    Techniques Used: Infection, Modification, Immunostaining, Plasmid Preparation, Generated

    4) Product Images from "An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2"

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258244

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.
    Figure Legend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Techniques Used: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).
    Figure Legend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Techniques Used: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.
    Figure Legend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Techniques Used: Binding Assay, Functional Assay, Derivative Assay

    5) Product Images from "Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity"

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

    Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    Figure Legend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P
    Figure Legend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Negative Control

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.
    Figure Legend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Techniques Used: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    6) Product Images from "SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.01283-20

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

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

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

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

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

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

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

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

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

    Techniques Used: Binding Assay

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

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

    7) Product Images from "An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2"

    Article Title: An effective, safe and cost-effective cell-based chimeric vaccine against SARS-CoV2

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258244

    The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.
    Figure Legend Snippet: The immunogenicity, efficacy and safety of C-Vac for SARS-CoV-2 infection. (A) Flow cytometry-based detection of MHC(HLA-A2)-peptide complex binding affinity in T2 cells. (B) The expression of antigens in 293T-based C-Vac is confirmed by Western Blot. N, 293T cells transfected with the plasmid expressing a full length N gene. (C D) Pseudovirus neutralization titers of hamster serum at day 7 after the first immunization and day 21 (boosted at day 14) after vaccination with 293T-based C-Vac, MiT C-Vac: Mitomycin C-treated C-Vac, Lys C-Vac: Lysed C-Vac. (E) Histological characteristics of hamster lung at day 7 after the first vaccination. Original magnification 200× (F) Allograft volume of transformed fibroblasts expressing RBD-truncated N protein in the Syrian hamsters immunized with different regime. 5×10 6 BHK21 cells expressing C-Vac antigen (RBD-Ntap) were subcutaneously injected into immunized hamsters for challenge at day 45 after boost, and the volume of allografts were measured at 14 days after inoculation of the BHK21 cells expressing RBD-Ntap into the immunized hamsters.

    Techniques Used: Infection, Flow Cytometry, Binding Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Neutralization, Transformation Assay, Injection

    The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).
    Figure Legend Snippet: The RBD domain of Spike is crucial for the SARS-CoV2 Vaccine. (A) The functional domain of SARS-CoV-2 spike protein. (B) Potential B cell antigen of RBD domain from SARS-CoV2 is predicted by Discotope software based on their 3D structure. (C) Potential linear B cell epitopes of SARS-CoV-2 full S protein are analysed with the IEDB database. (D) The location of potential antigens in RBD domain (SARS-CoV-2:red, SARS-CoV: Purple) and interaction model between RBD and ACE2 receptor (interface is marked yellow) are marked with Discovery Studio. (E) The expression of Spike and nucleocapsid with wild-type sequence in 293T cells are detected by Western Blot assay. (F) The expression of Spike and its derivatives with codon optimization (opt).

    Techniques Used: Functional Assay, Software, Expressing, Sequencing, Western Blot

    Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.
    Figure Legend Snippet: Construction of chimeric vaccine for SARS-CoV-2. (A) Potential B-cell epitopes of N protein is predicted by IEDB database. (B) Potential MHCI-binding peptides of N. (C) Functional domain of SARS-CoV N protein (Upper) and its antibody epitope map reported in previous study. (D) The skeleton of Chimeric Vaccine for SARS-CoV-2, RBD: spike RBD domain (306-541 aa), Ntap: T-cell-associated peptide of N (211-339 aa). (E) Characterization of SARS-CoV-2-derived protein and C-Vac antigen by SARS-CoV-2 antisera and commercial antibodies against SARS-CoV2 spike RBD or Nucleocapsid.

    Techniques Used: Binding Assay, Functional Assay, Derivative Assay

    8) Product Images from "Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity"

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

    Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    Figure Legend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P
    Figure Legend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Negative Control

    Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.
    Figure Legend Snippet: Pseudotyped SARS-CoV-2 virion neutralization activity of serum binding antibodies against S-RBD and N-protein. ( A ) Luminescence normalized to FBS+Virus control obtained from pseudovirus neutralization assay at 1:20 serum dilution. ( B ) Matched serological results for S-RBD at 1:100 serum dilution (top 2 panels) and 1:20 serum dilution (bottom 2 panels). Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. ( C ) Matched serological results for N-protein at 1:100 serum dilution and 1:20 serum dilution. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. Case numbers are color-coded: green: recovered, red: deceased, blue: hospitalized. Data ( A – C ) are reported as mean ± standard deviation (SD) of 3 technical replicates for each sample. ( D ) Heatmap depicting positive and negative categorization of the listed serum cases for each viral protein tested in serological and neutr3alization assays. Low titer positive as defined by detecting of binding antibodies shown in Figure 2, C and D , 1:20 titer.

    Techniques Used: Neutralization, Activity Assay, Binding Assay, Standard Deviation

    9) Product Images from "Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19"

    Article Title: Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.04.008

    Suppressive effects of dipyridamole (DIP) and chloroquine on SARS-CoV-2 replication in vitro . (A) Chemical structure of DIP. (B) Enzyme activity of Mpro in the presence of ascending concentrations of DIP. (C) Dose-dependent suppression of SARS-CoV-2 replication by DIP and chloroquine in vitro . Virus titers were measured by Foci forming assay, inhibition rates were performed by indirect immunoinfluscent assay, and calculated inhibition rates of different dosages of DIP or chloroquine were compared with virus control. P values were calculated by ANOVA.
    Figure Legend Snippet: Suppressive effects of dipyridamole (DIP) and chloroquine on SARS-CoV-2 replication in vitro . (A) Chemical structure of DIP. (B) Enzyme activity of Mpro in the presence of ascending concentrations of DIP. (C) Dose-dependent suppression of SARS-CoV-2 replication by DIP and chloroquine in vitro . Virus titers were measured by Foci forming assay, inhibition rates were performed by indirect immunoinfluscent assay, and calculated inhibition rates of different dosages of DIP or chloroquine were compared with virus control. P values were calculated by ANOVA.

    Techniques Used: In Vitro, Activity Assay, Inhibition

    10) Product Images from "Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery"

    Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

    Journal: bioRxiv

    doi: 10.1101/2020.08.02.233064

    Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.
    Figure Legend Snippet: Repurposed library screening for COVID-19 using phenomics A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.

    Techniques Used: Library Screening, Infection, Plasmid Preparation, Generated, Electric Cell-substrate Impedance Sensing, Concentration Assay, Staining

    SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors
    Figure Legend Snippet: SARS-CoV-2 infection model A . Quantification of active SARS-CoV-2 production over time in the indicated cell types using TCID50 measurement on Vero cells (n=2). B . Representative images of HRCE, Calu3 and Vero cells immunostained with SARS-CoV-2 nucleocapsid protein (pink) and modified cell paint dyes C . Infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. Of note, HRCE donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. Antibody stains were performed after the principal analysis concluded, and are therefore not represented in the primary dataset used for phenoprint evaluation and compound screening. D . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 and was selected for further investigation. Vero and Calu3 cells also demonstrated screenable phenoprints. E . Quantification of percentage of cells infected using nucleocapsid protein immunostaining in Calu3 cells at 96 hours post infection for key compounds F . Consistency of hit scores for selected compounds across HRCE donors and between cell types. G . Projections of compound response of JAK inhibitor and control compounds onto the perturbation vector generated in SARS-CoV-2-infected HRCE. H . Quantification of percent of cells infected using nucleocapsid protein immunostaining in HRCE cells at 96 hours post infection for JAK inhibitors

    Techniques Used: Infection, Modification, Immunostaining, Plasmid Preparation, Generated

    Related Articles

    Fluorescence:

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Neutralization:

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

    Microscopy:

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Incubation:

    Article Title: Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19
    Article Snippet: .. And then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Sino Biological, Inc., Beijing, China), followed by an HRP-labelled goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). .. The foci were visualized by TrueBlue™ Peroxidase Substrate (KPL, Gaithersburg, MD, USA), and counted with an ELISPOT reader (CTL, Shaker Heights, OH, USA).

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity
    Article Snippet: .. The membranes were blocked with 5% w/v milk in TBS/Tween 0.1% (TBS/T) and incubated with rabbit polyclonal anti-His (catalog 2365S, Cell Signaling Technology, 1:1000), rabbit polyclonal anti–SARS-CoV-2 Spike RBD (40592-T62, Sino Biological, 1:1000), or rabbit polyclonal anti–SARS-CoV2-Nucleocapsid protein (40588-T62, Sino Biological, 1:2000) antibodies in 2% BSA TBS/T (see ). .. Secondary antibodies used were donkey anti-rabbit HRP (ab16284, Abcam, 1:2000) in 2% BSA TBS/T.

    other:

    Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery
    Article Snippet: 40588-T62, 1:1000 dilution).

    Infection:

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

    Staining:

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2
    Article Snippet: .. Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008). .. Nuclei were counter-stained with diamidino-2-phenylindole (DAPI).

    Article Title: SARS-CoV-2 and Three Related Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig
    Article Snippet: .. The cells were then washed with serum-free medium and incubated in 150 μl of DMEM (2% FBS) at 37°C for an additional 24 h. The cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and sequentially stained with 1:200-diluted rabbit anti-SARS-CoV-2 nucleocapsid polyclonal antibody (Sino Biological, catalog no. 40588-T62) at 37°C for 30 min, 4 μg/ml of Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, catalog no. A-11011) at 37°C for 20 min, and 0.5 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Sigma-Aldrich, catalog no. D9542-5mg) at room temperature for 10 min. Stained cells were then examined under fluorescence microscope (IX73 microscope; Olympus). .. SARS-CoV-2 live virus neutralization by ACE2-Ig variants.

    Immunofluorescence:

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2
    Article Snippet: .. Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008). .. Nuclei were counter-stained with diamidino-2-phenylindole (DAPI).

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    ILRUN supresses <t>SARS-CoV-2</t> infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p
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    Sino Biological sars cov 2 s1 s2
    The levels of <t>SARS-CoV-2-reactive</t> to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p
    Sars Cov 2 S1 S2, 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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    sars cov 2 2019 ncov spike antibody rabbit pab  (Sino Biological)
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    Inhibitory activity of HP-OVA against <t>SARS-CoV-2</t> S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p
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    ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p

    Journal: bioRxiv

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

    doi: 10.1101/2020.11.13.381343

    Figure Lengend Snippet: ILRUN supresses SARS-CoV-2 infection and down-regulates host genes essential for SARS-CoV-2 entry. (A) Transcription profile of SARS-CoV-2 in Caco-2 cells transfected with 40 nM siNEG or siILRUN for 72 h at 6 h and 24 h post infection. (B) SARS-CoV-2 titres of supernatants from Caco-2 cells infected with SARS-CoV-2 (24 h, MOI 0.3) post-transfection with siRNAs (40 nM, 72 h) *p

    Article Snippet: Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection, Transfection

    Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p

    Journal: bioRxiv

    Article Title: ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

    doi: 10.1101/2020.11.13.381343

    Figure Lengend Snippet: Validation of ILRUN function and SARS-CoV-2 infection in Caco-2 cells. (A) ILRUN mRNA levels (2 −ΔΔCt relative to GAPDH ) in Caco-2 cells transfected with siRNAs (40 nM, 72 h) targeting ILRUN or a nontargeting control (siNEG). **p

    Article Snippet: Immunofluorescence and quantification of relative antigen stainingCaco-2 cells were fixed for 30 min in 4 % paraformaldehyde (PFA) and stained with a polyclonal antibody targeting the SARS-CoV-2 Nucleocapsid (N) protein (Sino Biological, catalogue number: 40588-T62, used at 1/2,000) for 1 h. Cells were subsequently stained with 1/1,000 dilution of an anti-rabbit AF488 antibody (Invitrogen catalogue number A11008).

    Techniques: Infection, Transfection

    The levels of SARS-CoV-2-reactive to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: The levels of SARS-CoV-2-reactive to S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic. The levels of a S1 + S2- and b nucleocapsid-reactive secretory IgM (SIgM)/IgM, IgG, and secretory IgA (SIgA)/IgA in human milk. Values are mean ± SD, n = 41 for women. Asterisks show statistically significant differences between variables (*** p

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques:

    Regression linear between antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic in 41 women. a Positive correlation of S1 + S2-reactive secretory IgA (SIgA)/IgA and S1 + S2-reactive secretory IgM (SIgM)/IgM. b Positive correlation between S1 + S2-reactive SIgA/IgA and nucleocapsid-reactive SIgA/IgA. c Positive correlation between nucleocapsid-reactive SIgA/IgA and SIgM/IgM. d Positive correlation between nucleocapsid-reactive SIgM/IgM and S1 + S2-reactive SIgM/IgM. Pearson correlation coefficients ( r ) were determined when p

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: Regression linear between antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic in 41 women. a Positive correlation of S1 + S2-reactive secretory IgA (SIgA)/IgA and S1 + S2-reactive secretory IgM (SIgM)/IgM. b Positive correlation between S1 + S2-reactive SIgA/IgA and nucleocapsid-reactive SIgA/IgA. c Positive correlation between nucleocapsid-reactive SIgA/IgA and SIgM/IgM. d Positive correlation between nucleocapsid-reactive SIgM/IgM and S1 + S2-reactive SIgM/IgM. Pearson correlation coefficients ( r ) were determined when p

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques:

    The levels of antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic (2020-HM) and 2 years prior this pandemic (2018-HM). Levels of S1 + S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in 2020-HM and 2018-HM. Levels of nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA in 2020-HM and 2018-HM. Values are mean ± SD, n = 41 for 2020-HM and n = 16 for 2018-HM. Asterisks show statistically significant differences between variables (* p

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: The levels of antibodies reactive to SARS-CoV-2 S1 and S2 subunits (S1 + S2) and nucleocapsid in human milk collected during the COVID-19 pandemic (2020-HM) and 2 years prior this pandemic (2018-HM). Levels of S1 + S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in 2020-HM and 2018-HM. Levels of nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA in 2020-HM and 2018-HM. Values are mean ± SD, n = 41 for 2020-HM and n = 16 for 2018-HM. Asterisks show statistically significant differences between variables (* p

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques:

    Percentage of detected antibodies reactive to S1 and S2 subunits (S1 + S2), and nucleocapsid from SARS-CoV-2 in human milk collected during the COVID-19 pandemic. S1+S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in human milk from 41 women. Nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA. Milk expression period was from 30/02/20 to 03/04/20 in the United States.

    Journal: Journal of Perinatology

    Article Title: Difference in levels of SARS-CoV-2 S1 and S2 subunits- and nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk

    doi: 10.1038/s41372-020-00805-w

    Figure Lengend Snippet: Percentage of detected antibodies reactive to S1 and S2 subunits (S1 + S2), and nucleocapsid from SARS-CoV-2 in human milk collected during the COVID-19 pandemic. S1+S2-reactive a secretory IgM (SIgM)/IgM, b IgG and c secretory IgA (SIgA)/IgA in human milk from 41 women. Nucleocapsid-reactive d SIgM/IgM, e IgG and f SIgA/IgA. Milk expression period was from 30/02/20 to 03/04/20 in the United States.

    Article Snippet: SARS-CoV-2 S1 + S2- and nucleocapsid-reactive antibodies The levels of SARS-CoV-2 S1 + S2 and nucleocapsid-reactive SIgM/IgM, IgG and SIgA/IgA were determined using ELISAs that were adapted from our previous publications [ , – ].

    Techniques: Expressing

    Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: Inhibitory activity of HP-OVA against SARS-CoV-2 S-mediated cell-cell fusion. Images were captured at 12 h after treatment with HP-OVA or OVA on SARS-CoV-2 S protein-mediated cell-cell fusion. The syncytia of Vero E6 cells (A) or Huh 7 cells (B) and HEK293T cells with SARS-CoV-2 overexpression are marked in the pictures. Representative results from three fields were selected randomly from each sample with scale bars of 50 μm (C, D) The number of syncytia was counted under an inverted fluorescence microscope, and the percentage of inhibition was calculated as described in the Methods. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Activity Assay, Over Expression, Fluorescence, Microscopy, Inhibition

    Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: Inhibition of HP-OVA on the infection with SARS-CoV-2 PsV and SARS-CoV PsV. Antiviral activity of HP-OVA against SARS-CoV-2 S PsV infection in 293T/ACE2 (A) or Vero E6 (B) target cells. Inhibition of single-round infection of SARS-CoV S PsV in 293T/ACE2 (C) and Vero E6 (D) cells. Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Inhibition, Infection, Activity Assay

    HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: HP-OVA binding to both SARS-CoV-2 S and ACE2 protein. Analysis of the expression of SARS-CoV-2 S (A) and ACE2 (B) in HEK-293T cells by western blot. The binding of HP-OVA to cells expressing SARS-CoV-2 S (C) or ACE2 (D) was assessed by flow cytometry. A representative flow histogram and quantification of the binding of HP-OVA to cells expressing SARS-CoV-2 S (E) or ACE2 (F) were shown. Data are presented as the mean ± SD (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

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

    The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: The interaction of HP-OVA with SARS-CoV-2 S and ACE2. The binding of OVA to SARS-CoV-2 spike (RBD), S2 and BSA protein was assessed by ELISA (A) . The binding of HP-OVA to SARS-CoV-2 spike (RBD), S2 and a negative control BSA protein was assessed by ELISA (B) . The binding ability of HP-OVA to ACE2 protein was assessed by ELISA (C) . Inhibition of the interaction between spike (RBD) and ACE2 proteins by HP-OVA, as determined by a competitive inhibition ELISA (D) . Data are presented as the mean ± SD of triplicate samples from a representative experiment (* p

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

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

    Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.

    Journal: Frontiers in Pharmacology

    Article Title: 3-Hydroxyphthalic Anhydride-Modified Chicken Ovalbumin as a Potential Candidate Inhibits SARS-CoV-2 Infection by Disrupting the Interaction of Spike Protein With Host ACE2 Receptor

    doi: 10.3389/fphar.2020.603830

    Figure Lengend Snippet: Schematic representation of the molecular mechanisms of HP-OVA against SARS-CoV-2 infection. HP-OVA binds to both the S protein of SARS-CoV-2 and host angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, and disrupts the S protein-ACE2 interaction, thereby exhibiting inhibitory activity against SARS-CoV-2 infection.

    Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA)ELISA was performed to identify the interaction of HP-OVA and the SARS-CoV-2 S protein (RBD) or ACE2 protein.

    Techniques: Infection, Functional Assay, Activity Assay

    A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: A model for structural rearrangements of SARS-Cov-2 S protein. (A) Structural changes independent of a target cell. We suggest that both the prefusion and postfusion spikes are present on the surface of mature virion and the ratio between them may vary (diagram of virion). The postfusion spikes on the virion are formed by S2 after S1 dissociates in the absence of ACE2. (B) ACE2-dependent structural rearrangements. Structural transition from the prefusion to postfusion conformation inducing membrane fusion likely proceeds stepwise as follows: 1) FPPR clamps down RBD through CTD1 in the prefusion S trimer, but it occasionally flips out of position and allows an RBD to sample the up conformation. 2) RBD binding to ACE2 creates a flexible FPPR that enables exposure of the S2’ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2’ site, and perhaps also the S1/S2 site, releases the structural constraints on the fusion peptide and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. 3) Formation of the long central three-stranded coiled-coil and folding back of HR2. 4) Formation of the postfusion structure of S2 that brings the two membranes together, facilitating formation of a fusion pore and viral entry.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Binding Assay

    Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Cryo-EM structure of the SARS-CoV-2 S2 in the postfusion conformation. (A) The structure of the S2 trimer was modeled based on a 3.3Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of the S2 trimer in the postfusion conformation shown in ribbon diagram. Various structural components in the color scheme shown in Fig. 1A include HR1, heptad repeat 1; CH, central helix region; CD, connector domain; and HR1, heptad repeat 2. The S2’ cleavage site is in a disordered loop between Ile770 and Thr912. A possible location of the S2 N-terminus (S1/S2 cleavage site) is also indicated. (C) A low resolution map showing the density pattern for 5 N-linked glycans, with almost equal spacing along the long axis.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques:

    Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Selected new features of the SARS-CoV-2 prefusion S trimer. (A) N-terminal segment of S protein. The N-terminus is at residue Gln14 after cleavage of the signal peptide. Cys15 forms a disulfide bond with Cys136. We observed good density for the N-linked glycan at Asn17. (B) A segment immediately downstream of the fusion peptide, while disordered in the stabilized soluble S ectodomain trimer structure, forms a tightly packed structure, designated FPPR for the fusion peptide proximal region, abutting CTD1. The newly identified FPPR structure would clash with CTD1 in the RBD up conformation. Various domains are shown in the color scheme in Fig. 2B . The structure of the soluble S trimer with one RBD in the up conformation (PDB ID: 6vyb) is shown in gray. In the box, a close-up view of the FPPR with adjacent fusion peptide in both surface representation and stick model. (C) The SARS-CoV-2 prefusion S trimer, viewed along the threefold axis, is superposed on the structure of the stabilized soluble S ectodomain trimer in the closed conformation with all three RBDs in the down conformation (PDB ID: 6vxx). While the S2 region is well aligned, there is a significant shift (e.g. ∼10Å between two Ala123 residues) in S1. (D) Impact of the proline mutations introduced at residues 986 and 987 to stabilize the prefusion conformation. K986P mutation removes a salt bridge between Lys986 of one protomer and either Asp426 or Asp427 of another protomer in the trimer interface.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Mutagenesis

    Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Preparation of a full-length SARS-CoV-2 spike protein. (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S) protein. Segments of S1 and S2 include: NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; FP, fusion peptide; FFPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor; CT, cytoplasmic tail; and tree-like symbols for glycans. A strep-tag was fused to the C-terminus of S protein by a flexible linker. (B) The purified S protein was resolved by gel-filtration chromatography on a Superose 6 column in the presence of detergent NP-40. The molecular weight standards include thyoglobulin (670 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). Three major peaks (peak I-III) contain the S protein. (C) Load sample and peak fractions from (B) were analyzed by Coomassie stained SDS-PAGE. Labeled bands were confirmed by western blot (S, S1 and S2) or protein sequencing (S2 and Cont; S and S1 bands did not gave any meaningful results probably due to a blocked N-terminus). Cont, copurified contaminating protein, identified as endoplasmic reticulum chaperone BiP precursor by N-terminal sequencing. Representative images and 2D averages by negative stain EM of three peak fractions are also shown. The box size of 2D averages is ∼510Å.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Expressing, Construct, Binding Assay, Strep-tag, Purification, Filtration, Chromatography, Molecular Weight, Staining, SDS Page, Labeling, Western Blot, Sequencing

    Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Journal: bioRxiv

    Article Title: Distinct conformational states of SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.16.099317

    Figure Lengend Snippet: Cryo-EM structure of the SARS-CoV-2 S protein in the prefusion conformation. (A) The structure of the S trimer was modeled based on a 3.1Å density map. Three protomers (A, B, C) are colored in green, blue and red, respectively. (B) Overall structure of S protein in the prefusion conformation shown in ribbon representation. Various structural components in the color scheme shown in Fig. 1A include NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; FP, fusion peptide; FPPR, fusion peptide proximal region; HR1, heptad repeat 1; CH, central helix region; and CD, connector domain. N-terminus, S1/S2 cleavage site and S2’ cleavage site are indicated.

    Article Snippet: Membranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature.

    Techniques: Binding Assay