sars cov 2 rbd  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD Antibody Mouse PAb
    Description:
    Produced in mice immunized with purified recombinant SARS CoV 2 2019 nCoV Spike RBD Protein Catalog 40592 V08H YP 009724390 1 Arg319 Phe541
    Catalog Number:
    40592-MP01
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    2019 nCoV
    Applications:
    ELISA
    Immunogen:
    Recombinant SARS-CoV-2 (2019-nCoV) Spike RBD-His Protein (Catalog#40592-V08H)
    Product Aliases:
    Anti-coronavirus spike Antibody, Anti-cov spike Antibody, Anti-ncov RBD Antibody, Anti-ncov s1 Antibody, Anti-ncov s2 Antibody, Anti-ncov spike Antibody, Anti-NCP-CoV RBD Antibody, Anti-NCP-CoV s1 Antibody, Anti-NCP-CoV s2 Antibody, Anti-NCP-CoV Spike Antibody, Anti-novel coronavirus RBD Antibody, Anti-novel coronavirus s1 Antibody, Anti-novel coronavirus s2 Antibody, Anti-novel coronavirus spike Antibody, Anti-RBD Antibody, Anti-S1 Antibody, Anti-S2 Antibody, Anti-Spike RBD Antibody
    Antibody Type:
    PAb
    Host:
    Mouse
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    Structured Review

    Sino Biological sars cov 2 rbd
    RU169 output clone diversity Using the <t>SARS-CoV-2</t> RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Produced in mice immunized with purified recombinant SARS CoV 2 2019 nCoV Spike RBD Protein Catalog 40592 V08H YP 009724390 1 Arg319 Phe541
    https://www.bioz.com/result/sars cov 2 rbd/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd - by Bioz Stars, 2021-06
    94/100 stars

    Images

    1) Product Images from "Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries"

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    Journal: bioRxiv

    doi: 10.1101/2020.07.27.224089

    RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Figure Legend Snippet: RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Techniques Used: FACS, Selection, Produced, Clone Assay

    BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.
    Figure Legend Snippet: BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Binding Assay

    Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.
    Figure Legend Snippet: Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Techniques Used: Clone Assay, In Vitro

    FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).
    Figure Legend Snippet: FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Techniques Used: FACS, Binding Assay, Labeling, Expressing, Clone Assay, Blocking Assay

    BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.
    Figure Legend Snippet: BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Techniques Used: Binding Assay

    Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .
    Figure Legend Snippet: Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Techniques Used: Cytometry, Inhibition, Fluorescence, Labeling

    2) Product Images from "Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients"

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

    Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.
    Figure Legend Snippet: Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.

    Techniques Used: Polymerase Chain Reaction

    AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.
    Figure Legend Snippet: AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Binding Assay, Derivative Assay

    Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.
    Figure Legend Snippet: Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Representative AIR array images (100 ms exposures) of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Figure 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.
    Figure Legend Snippet: Representative AIR array images (100 ms exposures) of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Figure 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.

    Techniques Used: Incubation

    Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.
    Figure Legend Snippet: Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    3) Product Images from "Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein"

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.0c01394

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.
    Figure Legend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Techniques Used: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.
    Figure Legend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Techniques Used: Flow Cytometry, Binding Assay

    Related Articles

    Selection:

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Article Snippet: .. SELEX Procedures We performed the aptamer selection procedure for SARS-CoV-2 RBD in a manner similar to our previous work. .. The initial ssDNA library consisted of a 40-nt randomized region and two flanking regions as a PCR primer (5′- ATCCAGAGTGACGCAGCA - 40N - TGGACACGGTGGCTTAGT-3′).

    other:

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein
    Article Snippet: By retaining the main motif of the full-length aptamers, we optimized two aptamers (with K d values of 5.8 nM and 19.9 nM) against SARS-CoV-2 RBD.

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients
    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Western Blot:

    Article Title: Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate
    Article Snippet: .. Western Blot AnalysisTo evaluate the SARS-CoV-2 RBD and S1 subunit proteins expression by rLS1-HN-RBD, and rLS1-S1-F recombinant viruses, Vero E6 cells were infected with the recombinant viruses and rLS1 at an MOI of 1. .. At 48 hpi the cells were harvested, lysed, and analyzed by Western blot.

    Expressing:

    Article Title: Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate
    Article Snippet: .. Western Blot AnalysisTo evaluate the SARS-CoV-2 RBD and S1 subunit proteins expression by rLS1-HN-RBD, and rLS1-S1-F recombinant viruses, Vero E6 cells were infected with the recombinant viruses and rLS1 at an MOI of 1. .. At 48 hpi the cells were harvested, lysed, and analyzed by Western blot.

    Recombinant:

    Article Title: Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate
    Article Snippet: .. Western Blot AnalysisTo evaluate the SARS-CoV-2 RBD and S1 subunit proteins expression by rLS1-HN-RBD, and rLS1-S1-F recombinant viruses, Vero E6 cells were infected with the recombinant viruses and rLS1 at an MOI of 1. .. At 48 hpi the cells were harvested, lysed, and analyzed by Western blot.

    Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2
    Article Snippet: The experiments included the following steps at 37°C: (1) equilibration (60 s); (2) activation of AR2G by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysucci-nimide (300 s); (3) immobilization of S1 protein onto sensors (100 s); (4) quenching with ethanolamine (300 s); (5) baseline in kinetics buffer (120 s); (6) association of antibodies for measurement of k on (300-600 s); and (7) dissociation of antibodies for measurement of k off (300-600 s). .. For measuring binding kinetics of single-domain antibodies with SARS-CoV-2 RBD, Avi-tagged recombinant RBD was biotinylated with the BirA biotinylation kit (Avidity), diluted in kinetics buffer and immobilized on streptavidin (SA) coated biosensors (Pall FortéBio) at ~50% of the sensor maximum binding capacity. ..

    Infection:

    Article Title: Development and pre-clinical evaluation of Newcastle disease virus-vectored SARS-CoV-2 intranasal vaccine candidate
    Article Snippet: .. Western Blot AnalysisTo evaluate the SARS-CoV-2 RBD and S1 subunit proteins expression by rLS1-HN-RBD, and rLS1-S1-F recombinant viruses, Vero E6 cells were infected with the recombinant viruses and rLS1 at an MOI of 1. .. At 48 hpi the cells were harvested, lysed, and analyzed by Western blot.

    Inhibition:

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries
    Article Snippet: Streptavidin biosensors (ForteBio, Cat: 18-5019) were loaded with AviTag™-biotinylated scFv or biotinylated SARS-CoV-2 S1 trimer (Acro Biosystems, Cat: S1N-C82E8), blocked with biotin, washed in PBS, and then associated with protein ligand in PBS. .. ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay. .. Biotinylated soluble ACE2 protein (Acro Biosystems, Cat: AC2-H82E6) was bound to MyOne Streptavidin C1 Dynabeads (0.33 mg ACE2 per mL Dynabeads) for 30 minutes, then the beads were magnetically purified, washed, and blocked with free biotin.

    Blocking Assay:

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries
    Article Snippet: Streptavidin biosensors (ForteBio, Cat: 18-5019) were loaded with AviTag™-biotinylated scFv or biotinylated SARS-CoV-2 S1 trimer (Acro Biosystems, Cat: S1N-C82E8), blocked with biotin, washed in PBS, and then associated with protein ligand in PBS. .. ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay. .. Biotinylated soluble ACE2 protein (Acro Biosystems, Cat: AC2-H82E6) was bound to MyOne Streptavidin C1 Dynabeads (0.33 mg ACE2 per mL Dynabeads) for 30 minutes, then the beads were magnetically purified, washed, and blocked with free biotin.

    Cell Culture:

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

    Binding Assay:

    Article Title: Identification of Human Single-Domain Antibodies against SARS-CoV-2
    Article Snippet: The experiments included the following steps at 37°C: (1) equilibration (60 s); (2) activation of AR2G by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysucci-nimide (300 s); (3) immobilization of S1 protein onto sensors (100 s); (4) quenching with ethanolamine (300 s); (5) baseline in kinetics buffer (120 s); (6) association of antibodies for measurement of k on (300-600 s); and (7) dissociation of antibodies for measurement of k off (300-600 s). .. For measuring binding kinetics of single-domain antibodies with SARS-CoV-2 RBD, Avi-tagged recombinant RBD was biotinylated with the BirA biotinylation kit (Avidity), diluted in kinetics buffer and immobilized on streptavidin (SA) coated biosensors (Pall FortéBio) at ~50% of the sensor maximum binding capacity. ..

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  • 97
    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Sars Cov2 S1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov2 s1/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov2 s1 - by Bioz Stars, 2021-06
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    Sino Biological sars cov 2 rbd
    RU169 output clone diversity Using the <t>SARS-CoV-2</t> RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Sars Cov 2 Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 rbd/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd - by Bioz Stars, 2021-06
    94/100 stars
      Buy from Supplier

    95
    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates <t>SARS-CoV-2</t> PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.
    Sars Cov 2 2019 Ncov Spike Rbd Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
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    99
    Sino Biological sars cov 2
    An openly available plasmid launched <t>SARS-CoV-2</t> RG system. (A) Schematic of the design and construction of a pCC1-4K-SARS-CoV-2-Wuhan-Hu-1 icDNA clone. Synthetic DNA fragments 1, 2, and 3 based on the SARS-CoV-2-Wuhan-Hu1 sequence were cloned into the pCC1 plasmids, and fragments 4 and 5 were cloned into high copy plasmid pUC57Kan by the gene synthesis company (Genscript). Fragments were designed to contain specific restriction cloning sites San Dl, Pac I, Mlu l, Bsu36 I, and Bam HI for cloning purposes. Sequences encoding for mCherry, ZsGreen, and NLuc markers were cloned in-frame to the C-terminus of the ORF7a protein via an FMDV 2A linker. (B) A summary of the passage (P) history of the extensively propagated wt 1 and wt 2 plasmid preps (expanded 6 times on solid agar and 5 times in liquid culture prior to being “grown up” in liquid culture for DNA extraction). (C) Summary plots of the number of reads mapping to the wt rescue plasmid and to the rescued viral genome. (D) Detection of SARS-CoV-2 N antigen in Vero E6 cells infected with RG-rescued SARS-CoV-2-Wuhan-Hu-1. Cells were not infected (Mock) or infected at MOI 1.0 for 48 h and then fixed and stained with mouse monoclonal anti-N antibody and counterstained with DAPI. Cells were imaged using a confocal microscope. Scale bar = 50 μm. (E) Titre of the mCherry virus (plaque forming units on Vero E6 cells) following propagation in Vero E6 cells (P3). (F) Detection of infected Vero E6 cells using the RG-rescued SARS-CoV-2-mCherry. Cells were infected for 48 h at MOI 1.0, then fixed, stained with anti-N antibody, and imaged as in (D). Scale bar = 50 μm. (G) Quantification of mCherry and N expression in Vero E6 cells infected with SARS-CoV-2-mCherry at an MOI of 0.1 for 48 h. Fixed and permeabilised cells were stained for N protein and with Hoechst 33342 (2 μg/ml). Cells were imaged using the Celigo Imaging Cytometer to identify the proportion of infected cells positive for mCherry and/or N protein. (H) Fixed plaque assays were scanned using a Celigo Imaging Cytometer (Nexcelom Bioscience) using the red channel to visualise mCherry. The cells were then subsequently stained with Coomassie staining solution and imaged again. (I) Detection of viral replication in infected Vero E6 cells using the RG-rescued SARS-CoV-2-NLuc. Cells were not infected (Mock) or infected at MOI 1.0 for 24 h, then lysed. NLuc activity was measured in the lysate using a luminometer. (J) Fluorescent plaques of the SARS-CoV-2-ZsGreen virus were visualised using the green channel as in panel H. (K) A schematic of the passage (P) history of the SARS-CoV-2 mCherry virus used to assess reporter stability. Each filled circle represents 1 day of propagation. (L) Plaque assays of passages 3 to 5 were scanned (Celigo) as in (H). The percentage of plaques visible following Coomassie staining that were mCherry-positive in the linear range of the dilution series (total plaque number in the range of 22 to 55 for each replicate) are plotted for each lineage at each passage. (M) Typical images of fluorescent plaques used for the quantification in (L) are shown. (N) A summary of the variation generated during the passage of the mCherry virus summarised in panel K. The variation is detailed in S1 Table . (O) The percentage of the viral swarm displaying all variants that exceed 25% of the swarm at any time point is shown. The low-level deletion of the furin cleavage site is also highlighted for interest. The data underlying Fig 1E, 1G, 1I and 1L may be found in S1 Data . FMDV, foot-and-mouth disease virus; icDNA, infectious cDNA; MOI, multiplicity of infection; NLuc, Nanoluciferase; RG, reverse genetics; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; wt, wild-type.
    Sars Cov 2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 2 - by Bioz Stars, 2021-06
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    Image Search Results


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

    Journal: Small Methods

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

    doi: 10.1002/smtd.202001031

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

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

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

    RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: FACS, Selection, Produced, Clone Assay

    BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Clone Assay, Plasmid Preparation, Purification, Binding Assay

    Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Clone Assay, In Vitro

    FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: FACS, Binding Assay, Labeling, Expressing, Clone Assay, Blocking Assay

    BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Binding Assay

    Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Cytometry, Inhibition, Fluorescence, Labeling

    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Labeling, Förster Resonance Energy Transfer

    An openly available plasmid launched SARS-CoV-2 RG system. (A) Schematic of the design and construction of a pCC1-4K-SARS-CoV-2-Wuhan-Hu-1 icDNA clone. Synthetic DNA fragments 1, 2, and 3 based on the SARS-CoV-2-Wuhan-Hu1 sequence were cloned into the pCC1 plasmids, and fragments 4 and 5 were cloned into high copy plasmid pUC57Kan by the gene synthesis company (Genscript). Fragments were designed to contain specific restriction cloning sites San Dl, Pac I, Mlu l, Bsu36 I, and Bam HI for cloning purposes. Sequences encoding for mCherry, ZsGreen, and NLuc markers were cloned in-frame to the C-terminus of the ORF7a protein via an FMDV 2A linker. (B) A summary of the passage (P) history of the extensively propagated wt 1 and wt 2 plasmid preps (expanded 6 times on solid agar and 5 times in liquid culture prior to being “grown up” in liquid culture for DNA extraction). (C) Summary plots of the number of reads mapping to the wt rescue plasmid and to the rescued viral genome. (D) Detection of SARS-CoV-2 N antigen in Vero E6 cells infected with RG-rescued SARS-CoV-2-Wuhan-Hu-1. Cells were not infected (Mock) or infected at MOI 1.0 for 48 h and then fixed and stained with mouse monoclonal anti-N antibody and counterstained with DAPI. Cells were imaged using a confocal microscope. Scale bar = 50 μm. (E) Titre of the mCherry virus (plaque forming units on Vero E6 cells) following propagation in Vero E6 cells (P3). (F) Detection of infected Vero E6 cells using the RG-rescued SARS-CoV-2-mCherry. Cells were infected for 48 h at MOI 1.0, then fixed, stained with anti-N antibody, and imaged as in (D). Scale bar = 50 μm. (G) Quantification of mCherry and N expression in Vero E6 cells infected with SARS-CoV-2-mCherry at an MOI of 0.1 for 48 h. Fixed and permeabilised cells were stained for N protein and with Hoechst 33342 (2 μg/ml). Cells were imaged using the Celigo Imaging Cytometer to identify the proportion of infected cells positive for mCherry and/or N protein. (H) Fixed plaque assays were scanned using a Celigo Imaging Cytometer (Nexcelom Bioscience) using the red channel to visualise mCherry. The cells were then subsequently stained with Coomassie staining solution and imaged again. (I) Detection of viral replication in infected Vero E6 cells using the RG-rescued SARS-CoV-2-NLuc. Cells were not infected (Mock) or infected at MOI 1.0 for 24 h, then lysed. NLuc activity was measured in the lysate using a luminometer. (J) Fluorescent plaques of the SARS-CoV-2-ZsGreen virus were visualised using the green channel as in panel H. (K) A schematic of the passage (P) history of the SARS-CoV-2 mCherry virus used to assess reporter stability. Each filled circle represents 1 day of propagation. (L) Plaque assays of passages 3 to 5 were scanned (Celigo) as in (H). The percentage of plaques visible following Coomassie staining that were mCherry-positive in the linear range of the dilution series (total plaque number in the range of 22 to 55 for each replicate) are plotted for each lineage at each passage. (M) Typical images of fluorescent plaques used for the quantification in (L) are shown. (N) A summary of the variation generated during the passage of the mCherry virus summarised in panel K. The variation is detailed in S1 Table . (O) The percentage of the viral swarm displaying all variants that exceed 25% of the swarm at any time point is shown. The low-level deletion of the furin cleavage site is also highlighted for interest. The data underlying Fig 1E, 1G, 1I and 1L may be found in S1 Data . FMDV, foot-and-mouth disease virus; icDNA, infectious cDNA; MOI, multiplicity of infection; NLuc, Nanoluciferase; RG, reverse genetics; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; wt, wild-type.

    Journal: PLoS Biology

    Article Title: A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

    doi: 10.1371/journal.pbio.3001091

    Figure Lengend Snippet: An openly available plasmid launched SARS-CoV-2 RG system. (A) Schematic of the design and construction of a pCC1-4K-SARS-CoV-2-Wuhan-Hu-1 icDNA clone. Synthetic DNA fragments 1, 2, and 3 based on the SARS-CoV-2-Wuhan-Hu1 sequence were cloned into the pCC1 plasmids, and fragments 4 and 5 were cloned into high copy plasmid pUC57Kan by the gene synthesis company (Genscript). Fragments were designed to contain specific restriction cloning sites San Dl, Pac I, Mlu l, Bsu36 I, and Bam HI for cloning purposes. Sequences encoding for mCherry, ZsGreen, and NLuc markers were cloned in-frame to the C-terminus of the ORF7a protein via an FMDV 2A linker. (B) A summary of the passage (P) history of the extensively propagated wt 1 and wt 2 plasmid preps (expanded 6 times on solid agar and 5 times in liquid culture prior to being “grown up” in liquid culture for DNA extraction). (C) Summary plots of the number of reads mapping to the wt rescue plasmid and to the rescued viral genome. (D) Detection of SARS-CoV-2 N antigen in Vero E6 cells infected with RG-rescued SARS-CoV-2-Wuhan-Hu-1. Cells were not infected (Mock) or infected at MOI 1.0 for 48 h and then fixed and stained with mouse monoclonal anti-N antibody and counterstained with DAPI. Cells were imaged using a confocal microscope. Scale bar = 50 μm. (E) Titre of the mCherry virus (plaque forming units on Vero E6 cells) following propagation in Vero E6 cells (P3). (F) Detection of infected Vero E6 cells using the RG-rescued SARS-CoV-2-mCherry. Cells were infected for 48 h at MOI 1.0, then fixed, stained with anti-N antibody, and imaged as in (D). Scale bar = 50 μm. (G) Quantification of mCherry and N expression in Vero E6 cells infected with SARS-CoV-2-mCherry at an MOI of 0.1 for 48 h. Fixed and permeabilised cells were stained for N protein and with Hoechst 33342 (2 μg/ml). Cells were imaged using the Celigo Imaging Cytometer to identify the proportion of infected cells positive for mCherry and/or N protein. (H) Fixed plaque assays were scanned using a Celigo Imaging Cytometer (Nexcelom Bioscience) using the red channel to visualise mCherry. The cells were then subsequently stained with Coomassie staining solution and imaged again. (I) Detection of viral replication in infected Vero E6 cells using the RG-rescued SARS-CoV-2-NLuc. Cells were not infected (Mock) or infected at MOI 1.0 for 24 h, then lysed. NLuc activity was measured in the lysate using a luminometer. (J) Fluorescent plaques of the SARS-CoV-2-ZsGreen virus were visualised using the green channel as in panel H. (K) A schematic of the passage (P) history of the SARS-CoV-2 mCherry virus used to assess reporter stability. Each filled circle represents 1 day of propagation. (L) Plaque assays of passages 3 to 5 were scanned (Celigo) as in (H). The percentage of plaques visible following Coomassie staining that were mCherry-positive in the linear range of the dilution series (total plaque number in the range of 22 to 55 for each replicate) are plotted for each lineage at each passage. (M) Typical images of fluorescent plaques used for the quantification in (L) are shown. (N) A summary of the variation generated during the passage of the mCherry virus summarised in panel K. The variation is detailed in S1 Table . (O) The percentage of the viral swarm displaying all variants that exceed 25% of the swarm at any time point is shown. The low-level deletion of the furin cleavage site is also highlighted for interest. The data underlying Fig 1E, 1G, 1I and 1L may be found in S1 Data . FMDV, foot-and-mouth disease virus; icDNA, infectious cDNA; MOI, multiplicity of infection; NLuc, Nanoluciferase; RG, reverse genetics; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; wt, wild-type.

    Article Snippet: ImmunofluorescenceCells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection.

    Techniques: Plasmid Preparation, Sequencing, Clone Assay, DNA Extraction, Infection, Staining, Microscopy, Expressing, Imaging, Cytometry, Activity Assay, Generated

    Production and validation of a near-comprehensive panel of openly available SARS-CoV-2 and coronavirus antibodies. (A) Schematic representation of the SARS-CoV-2 and other coronavirus-processed proteins for which antibody synthesis and validation is described in this study. All antibodies (and corresponding proteins and cDNAs) listed are available upon request at https://mrcppu-covid.bio/ . Accession numbers for the sequences utilised can be found in S2 Table and at https://mrcppu-covid.bio/ . (B) A schematic illustrating the method of production for the antibodies shown in (A), utilising N (nucleocapsid) as an example. Each cDNA encoding a coronavirus protein was cloned into both pGex (carrying a GST tag) and pMex (MBP tag) plasmids, in order to yield corresponding GST- and MBP-tagged viral proteins. Sheep were subsequently immunised by utilising the GST-tagged proteins as antigens. At day 7 postimmunisation, serum was harvested and antibodies were affinity purified using the corresponding MBP-tagged protein. Additional inoculations (up to 5 in total) occurred 28 days apart. Exceptions to this method occurred for SARS-CoV-2 ORF7a, S, and S-RBD antibodies, where the MBP-tagged proteins were used as antigens, and after harvest, serum was again purified against the MBP-tagged protein, followed by depletion of the antibody against MBP. The SARS-CoV-2 S antibody was purified using protein G Sepharose chromatography. (C) IF validation for three of the antibodies (against nonstructural nsp2, structural N, and accessory ORF3a proteins) shown in (A) was conducted in Vero E6 cells that were uninfected (mock) or infected with the SARS-CoV-2 England-02 virus at an MOI of 0.1 for 48 h prior to fixation, permeabilization, and staining. IF validation for the remaining antibodies in (A) is shown in S2 Fig . (D) WB validation for the 3 antibodies shown in (C) was conducted using Vero E6 cells that were uninfected (mock) or infected with England-02 virus at an MOI of 0.1 or 1 (as indicated) for 72 h followed by WB analysis of whole cell lysates. WB validation for the remaining antibodies in (A) is shown in S3 and S4 Figs. (E) IP validation for the 3 antibodies shown in (C) was conducted using Vero E6 cells that were uninfected (mock) or infected with England-02 virus at an MOI of 0.1 for 3 days. IP validation for the remaining antibodies in (A) is shown in S4 and S5 Figs. (F) Immunostaining of cells either uninfected (mock) or infected with the RG-rescued SARS-CoV-2 and its mCherry and NLuc derivatives. Cells were stained using the N antibody utilised in (C–E) and imaged using a Celigo imaging cytometer. (G) WB validation of Vero E6 cells infected for 48 h with the RG viruses shown from Fig 1A for 48 h, using the 3 antibodies shown in (C–E). GST, glutathione S-transferase; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; MBP, maltose-binding protein; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; MOI, multiplicity of infection; NLuc, Nanoluciferase; RG, reverse genetics; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WB, western blotting; wt, wild-type.

    Journal: PLoS Biology

    Article Title: A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

    doi: 10.1371/journal.pbio.3001091

    Figure Lengend Snippet: Production and validation of a near-comprehensive panel of openly available SARS-CoV-2 and coronavirus antibodies. (A) Schematic representation of the SARS-CoV-2 and other coronavirus-processed proteins for which antibody synthesis and validation is described in this study. All antibodies (and corresponding proteins and cDNAs) listed are available upon request at https://mrcppu-covid.bio/ . Accession numbers for the sequences utilised can be found in S2 Table and at https://mrcppu-covid.bio/ . (B) A schematic illustrating the method of production for the antibodies shown in (A), utilising N (nucleocapsid) as an example. Each cDNA encoding a coronavirus protein was cloned into both pGex (carrying a GST tag) and pMex (MBP tag) plasmids, in order to yield corresponding GST- and MBP-tagged viral proteins. Sheep were subsequently immunised by utilising the GST-tagged proteins as antigens. At day 7 postimmunisation, serum was harvested and antibodies were affinity purified using the corresponding MBP-tagged protein. Additional inoculations (up to 5 in total) occurred 28 days apart. Exceptions to this method occurred for SARS-CoV-2 ORF7a, S, and S-RBD antibodies, where the MBP-tagged proteins were used as antigens, and after harvest, serum was again purified against the MBP-tagged protein, followed by depletion of the antibody against MBP. The SARS-CoV-2 S antibody was purified using protein G Sepharose chromatography. (C) IF validation for three of the antibodies (against nonstructural nsp2, structural N, and accessory ORF3a proteins) shown in (A) was conducted in Vero E6 cells that were uninfected (mock) or infected with the SARS-CoV-2 England-02 virus at an MOI of 0.1 for 48 h prior to fixation, permeabilization, and staining. IF validation for the remaining antibodies in (A) is shown in S2 Fig . (D) WB validation for the 3 antibodies shown in (C) was conducted using Vero E6 cells that were uninfected (mock) or infected with England-02 virus at an MOI of 0.1 or 1 (as indicated) for 72 h followed by WB analysis of whole cell lysates. WB validation for the remaining antibodies in (A) is shown in S3 and S4 Figs. (E) IP validation for the 3 antibodies shown in (C) was conducted using Vero E6 cells that were uninfected (mock) or infected with England-02 virus at an MOI of 0.1 for 3 days. IP validation for the remaining antibodies in (A) is shown in S4 and S5 Figs. (F) Immunostaining of cells either uninfected (mock) or infected with the RG-rescued SARS-CoV-2 and its mCherry and NLuc derivatives. Cells were stained using the N antibody utilised in (C–E) and imaged using a Celigo imaging cytometer. (G) WB validation of Vero E6 cells infected for 48 h with the RG viruses shown from Fig 1A for 48 h, using the 3 antibodies shown in (C–E). GST, glutathione S-transferase; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; MBP, maltose-binding protein; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; MOI, multiplicity of infection; NLuc, Nanoluciferase; RG, reverse genetics; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WB, western blotting; wt, wild-type.

    Article Snippet: ImmunofluorescenceCells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection.

    Techniques: Clone Assay, Affinity Purification, Purification, Chromatography, Infection, Staining, Western Blot, Immunostaining, Imaging, Cytometry, Immunofluorescence, Immunoprecipitation, Binding Assay

    Toolkit of SARS-CoV-2 clinical isolates. (A) A comparison of plaque phenotypes in VAT cells from the 3 SARS-CoV-2 viruses isolated in this study and the England-02 SARS-CoV-2 virus. (B) A comparison of viral titers (in pfu/ml) of the 3 SARS-CoV-2 viruses isolated in this study and the England-02 SARS-CoV-2 virus in AA, AAT, Vero E6 (E6), VA, and VAT cells. All cells with exogenous ACE2 or TMPRSS2 were transduced with the lentiviruses described in Fig 4 . (C) Quantification of plaque area (in mm 2 ) was calculated in ImageJ (plaques not overlapping other plaques or the side of the well) from 4 replicates of the data presented in (A) and (B). Significance was determined using a Mann–Whitney U test. (D) A table showing the amino acid substitutions present in England-02 and 3 SARS-CoV-2 viruses isolated from clinical samples in this study, relative to Wuhan-Hu-1 (NC_045512). CVR-GLA-1 (MT882022) was isolated from sputum (CVR837 [EPI_ISL_461705]), CVR-GLA-2 (MT906650) was isolated from sputum (CVR2224 [EPI_ISL_448167], and CVR-GLA-3 (MT906649) was isolated from bronchoalveolar lavage (CVR3899_BAL [EPI_ISL_490695]). (E) IF of the 3 SARS-CoV-2 viruses isolated in this study and the England-02 SARS-CoV-2 virus in AAT cells (at MOI 0.01 for 48 h), using ORF3a, N, and nsp2 antibodies, as in Fig 2 . The data underlying Fig 5B and 5C may be found in S1 Data . AA, A549-ACE2; AAT, A549-ACE2-TMPRSS2; ACE2, angiotensin-converting enzyme 2; IF, immunofluorescence; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VA, Vero E6-ACE2; VAT, Vero E6-ACE2-TMPRSS2.

    Journal: PLoS Biology

    Article Title: A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

    doi: 10.1371/journal.pbio.3001091

    Figure Lengend Snippet: Toolkit of SARS-CoV-2 clinical isolates. (A) A comparison of plaque phenotypes in VAT cells from the 3 SARS-CoV-2 viruses isolated in this study and the England-02 SARS-CoV-2 virus. (B) A comparison of viral titers (in pfu/ml) of the 3 SARS-CoV-2 viruses isolated in this study and the England-02 SARS-CoV-2 virus in AA, AAT, Vero E6 (E6), VA, and VAT cells. All cells with exogenous ACE2 or TMPRSS2 were transduced with the lentiviruses described in Fig 4 . (C) Quantification of plaque area (in mm 2 ) was calculated in ImageJ (plaques not overlapping other plaques or the side of the well) from 4 replicates of the data presented in (A) and (B). Significance was determined using a Mann–Whitney U test. (D) A table showing the amino acid substitutions present in England-02 and 3 SARS-CoV-2 viruses isolated from clinical samples in this study, relative to Wuhan-Hu-1 (NC_045512). CVR-GLA-1 (MT882022) was isolated from sputum (CVR837 [EPI_ISL_461705]), CVR-GLA-2 (MT906650) was isolated from sputum (CVR2224 [EPI_ISL_448167], and CVR-GLA-3 (MT906649) was isolated from bronchoalveolar lavage (CVR3899_BAL [EPI_ISL_490695]). (E) IF of the 3 SARS-CoV-2 viruses isolated in this study and the England-02 SARS-CoV-2 virus in AAT cells (at MOI 0.01 for 48 h), using ORF3a, N, and nsp2 antibodies, as in Fig 2 . The data underlying Fig 5B and 5C may be found in S1 Data . AA, A549-ACE2; AAT, A549-ACE2-TMPRSS2; ACE2, angiotensin-converting enzyme 2; IF, immunofluorescence; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VA, Vero E6-ACE2; VAT, Vero E6-ACE2-TMPRSS2.

    Article Snippet: ImmunofluorescenceCells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection.

    Techniques: Isolation, Transduction, MANN-WHITNEY, Immunofluorescence, Infection

    Sequence changes observed in SARS-CoV-2 CVR-GLA-1 following in vitro propagation. (A) A schematic of the passage history of the SARS-CoV-2 CVR-GLA-1 virus in Vero E6 and AAT cells. Each filled circle represents 1 day of propagation. (B) A summary of the variation generated during the passage of the CVR-GLA-1 virus indicated in panel A. The variation is detailed in S1 Table . The percentage of the viral swarm displaying all variants that exceed 5% or 25% of the swarm at any time point is shown. The percentage occurrence of several deletion mutants is highlighted for interest. AAT, A549-ACE2-TMPRSS2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

    Journal: PLoS Biology

    Article Title: A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

    doi: 10.1371/journal.pbio.3001091

    Figure Lengend Snippet: Sequence changes observed in SARS-CoV-2 CVR-GLA-1 following in vitro propagation. (A) A schematic of the passage history of the SARS-CoV-2 CVR-GLA-1 virus in Vero E6 and AAT cells. Each filled circle represents 1 day of propagation. (B) A summary of the variation generated during the passage of the CVR-GLA-1 virus indicated in panel A. The variation is detailed in S1 Table . The percentage of the viral swarm displaying all variants that exceed 5% or 25% of the swarm at any time point is shown. The percentage occurrence of several deletion mutants is highlighted for interest. AAT, A549-ACE2-TMPRSS2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

    Article Snippet: ImmunofluorescenceCells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection.

    Techniques: Sequencing, In Vitro, Generated

    Usage of our toolkit antibodies to demonstrate or confirm SARS-CoV-2 protein interactions. (A) A co-IP was performed using lysates from Vero E6 cells infected with England-02 at MOI 0.1 for 3 days. Using the specific anti-ORF3a antibody described herein, SARS-CoV-2 ORF3a was immunoprecipitated (alongside a preimmune IgG control), and the immune complexes were western blotted for the presence of SARS-CoV-2 spike (S) and ORF3a. (B) As in (A), SARS-CoV-2 nsp13 (or IgG control) was immunoprecipitated and the immune complexes were probed for nsp13 and nsp11/12 by WB. (C) As in (A, B), SARS-CoV-2 N (or IgG control) was immunoprecipitated and the immune complexes were probed for matrix (M) and N by WB. co-IP, co-immunoprecipitation; IB, immunoblotting; IgG, immunoglobulin G; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WB, western blotting.

    Journal: PLoS Biology

    Article Title: A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

    doi: 10.1371/journal.pbio.3001091

    Figure Lengend Snippet: Usage of our toolkit antibodies to demonstrate or confirm SARS-CoV-2 protein interactions. (A) A co-IP was performed using lysates from Vero E6 cells infected with England-02 at MOI 0.1 for 3 days. Using the specific anti-ORF3a antibody described herein, SARS-CoV-2 ORF3a was immunoprecipitated (alongside a preimmune IgG control), and the immune complexes were western blotted for the presence of SARS-CoV-2 spike (S) and ORF3a. (B) As in (A), SARS-CoV-2 nsp13 (or IgG control) was immunoprecipitated and the immune complexes were probed for nsp13 and nsp11/12 by WB. (C) As in (A, B), SARS-CoV-2 N (or IgG control) was immunoprecipitated and the immune complexes were probed for matrix (M) and N by WB. co-IP, co-immunoprecipitation; IB, immunoblotting; IgG, immunoglobulin G; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WB, western blotting.

    Article Snippet: ImmunofluorescenceCells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection.

    Techniques: Co-Immunoprecipitation Assay, Infection, Immunoprecipitation, Western Blot

    Conventional cell lines modified to express ACE2 and TMPRSS2 lentiviruses have utility in SARS-CoV-2 phenotypic assays. (A) A schematic illustrating the LV-ACE2 and LV-TMPRSS2 lentiviruses that have been used to transduce multiple cell lines. The corresponding abbreviations of the modified lines are also shown. (B) The abundance of ACE2 and TMPRSS2 in Vero E6 cells, A549 cells and derivatives modified to express exogenous ACE2 and/or TMPRSS2 was assessed by WB. Permissive Calu-3 and Caco-2 samples were included for reference. Two actin blots are presented as different samples of equivalent cells were used to stain for ACE2 and TMPRSS2. (C) ACE2 intensity values from confocal microscopy images of ACE2 and Hoechst-stained Vero E6 cells and VA, VT, and VAT derivatives were measured using Cell Profiler ( cellprofiler.org ). (D) Randomly selected confocal microscopy images used for the quantification in (C) are shown. (E) As in panel C, using stained A549 cells, as well as AA and AAT cell derivatives. (F) Randomly selected confocal microscopy images used for the quantification in (E) are shown. (G) The plaque phenotype produced by the same preparation of SARS-CoV-2 England-02 on Vero E6 cells and VA, VT, and VAT derivatives. (H) As in (G), indicating the titre (in pfu/ml) of the same preparation of SARS-CoV-2 England-02 in cells with or without exogenous ACE2 and/or TMPRSS2. (I) As in panel G, AA and AAT derivatives of A549 cells were infected with SARS-CoV-2 England-02 to observe the plaque phenotype. (J) An example of a phenotypic well clearance/monolayer integrity assay used to assess the anti-SARS-CoV-2 activity of various compounds (nafamostat, apilimod, EIDD_2801, or remdesivir). The cells were treated with 2-fold serially diluted compound (10 μM to 20 nM) before being mock infected or infected with SARS-CoV-2. At 72 h postinfection, the monolayers were fixed and Coomassie-stained before scanning using a Celigo imaging cytometer. (K) As in panel J, well clearance assays in AA and AAT cells using SARS-CoV-2 England-02 are shown. (L) Quantification of the anti-SARS-CoV-2 activity of apilimod in AA and AAT cells. The mean and standard error of 4 replicates are plotted. (M) Dose response curve of remdesivir using the well-clearance assay in AA cells (as in panel K) multiplexed with a dead cell protease toxicity assay. The mean and standard error of 4 replicates are plotted. (N) as in panel M using AAT cells. The data underlying Fig 4C, 4E, 4H, 4L, 4M and 4N may be found in S1 Data . AA, A549-ACE2; AAT, A549-ACE2-TMPRSS2; ACE2, angiotensin-converting enzyme 2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VA, Vero E6-ACE2; VAT, Vero E6-ACE2-TMPRSS2; VT, Vero E6-TMPRSS2; WB, western blotting.

    Journal: PLoS Biology

    Article Title: A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

    doi: 10.1371/journal.pbio.3001091

    Figure Lengend Snippet: Conventional cell lines modified to express ACE2 and TMPRSS2 lentiviruses have utility in SARS-CoV-2 phenotypic assays. (A) A schematic illustrating the LV-ACE2 and LV-TMPRSS2 lentiviruses that have been used to transduce multiple cell lines. The corresponding abbreviations of the modified lines are also shown. (B) The abundance of ACE2 and TMPRSS2 in Vero E6 cells, A549 cells and derivatives modified to express exogenous ACE2 and/or TMPRSS2 was assessed by WB. Permissive Calu-3 and Caco-2 samples were included for reference. Two actin blots are presented as different samples of equivalent cells were used to stain for ACE2 and TMPRSS2. (C) ACE2 intensity values from confocal microscopy images of ACE2 and Hoechst-stained Vero E6 cells and VA, VT, and VAT derivatives were measured using Cell Profiler ( cellprofiler.org ). (D) Randomly selected confocal microscopy images used for the quantification in (C) are shown. (E) As in panel C, using stained A549 cells, as well as AA and AAT cell derivatives. (F) Randomly selected confocal microscopy images used for the quantification in (E) are shown. (G) The plaque phenotype produced by the same preparation of SARS-CoV-2 England-02 on Vero E6 cells and VA, VT, and VAT derivatives. (H) As in (G), indicating the titre (in pfu/ml) of the same preparation of SARS-CoV-2 England-02 in cells with or without exogenous ACE2 and/or TMPRSS2. (I) As in panel G, AA and AAT derivatives of A549 cells were infected with SARS-CoV-2 England-02 to observe the plaque phenotype. (J) An example of a phenotypic well clearance/monolayer integrity assay used to assess the anti-SARS-CoV-2 activity of various compounds (nafamostat, apilimod, EIDD_2801, or remdesivir). The cells were treated with 2-fold serially diluted compound (10 μM to 20 nM) before being mock infected or infected with SARS-CoV-2. At 72 h postinfection, the monolayers were fixed and Coomassie-stained before scanning using a Celigo imaging cytometer. (K) As in panel J, well clearance assays in AA and AAT cells using SARS-CoV-2 England-02 are shown. (L) Quantification of the anti-SARS-CoV-2 activity of apilimod in AA and AAT cells. The mean and standard error of 4 replicates are plotted. (M) Dose response curve of remdesivir using the well-clearance assay in AA cells (as in panel K) multiplexed with a dead cell protease toxicity assay. The mean and standard error of 4 replicates are plotted. (N) as in panel M using AAT cells. The data underlying Fig 4C, 4E, 4H, 4L, 4M and 4N may be found in S1 Data . AA, A549-ACE2; AAT, A549-ACE2-TMPRSS2; ACE2, angiotensin-converting enzyme 2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VA, Vero E6-ACE2; VAT, Vero E6-ACE2-TMPRSS2; VT, Vero E6-TMPRSS2; WB, western blotting.

    Article Snippet: ImmunofluorescenceCells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection.

    Techniques: Modification, Transduction, Western Blot, Staining, Confocal Microscopy, Produced, Infection, Integrity Assay, Activity Assay, Imaging, Cytometry