mouse igg1 fc domain  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD mFc Recombinant Protein HPLC verified COVID 19 Spike RBD Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of mouse IgG1 at the C terminus
    Catalog Number:
    40592-V05H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological mouse igg1 fc domain
    Functional assays from single antigen-reactive B cells. a. Schematic of detection of antigen-specific antibody . Biotinylated antigen (dark grey) was coupled to a streptavidin-conjugated polystyrene bead (light grey). Antibodies (blue) are secreted by single B cells loaded into individual NanoPens on the Berkeley Lights Beacon optofluidic device. Antibody binding to antigen was detected with a fluorescent anti-human <t>IgG</t> secondary Ab (black). b. Left : Schematic of fluorescing beads in the channel above a pen containing an individual B cell indicates antigen-specific reactivity. Top right : False-color still image of positive wells with B cells secreting S2P ecto -reactive antibodies. Reactive antibody diffusing out of a pen is visualized as a plume of fluorescence. Bottom right : False-color still image of positive wells with B cells secreting RBD-mFc-reactive antibodies. c . Representative images of RBD-mFc reactive clones.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of mouse IgG1 at the C terminus
    https://www.bioz.com/result/mouse igg1 fc domain/product/Sino Biological
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    mouse igg1 fc domain - by Bioz Stars, 2021-07
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    Images

    1) Product Images from "Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein"

    Article Title: Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

    Journal: bioRxiv

    doi: 10.1101/2020.05.12.091462

    Functional assays from single antigen-reactive B cells. a. Schematic of detection of antigen-specific antibody . Biotinylated antigen (dark grey) was coupled to a streptavidin-conjugated polystyrene bead (light grey). Antibodies (blue) are secreted by single B cells loaded into individual NanoPens on the Berkeley Lights Beacon optofluidic device. Antibody binding to antigen was detected with a fluorescent anti-human IgG secondary Ab (black). b. Left : Schematic of fluorescing beads in the channel above a pen containing an individual B cell indicates antigen-specific reactivity. Top right : False-color still image of positive wells with B cells secreting S2P ecto -reactive antibodies. Reactive antibody diffusing out of a pen is visualized as a plume of fluorescence. Bottom right : False-color still image of positive wells with B cells secreting RBD-mFc-reactive antibodies. c . Representative images of RBD-mFc reactive clones.
    Figure Legend Snippet: Functional assays from single antigen-reactive B cells. a. Schematic of detection of antigen-specific antibody . Biotinylated antigen (dark grey) was coupled to a streptavidin-conjugated polystyrene bead (light grey). Antibodies (blue) are secreted by single B cells loaded into individual NanoPens on the Berkeley Lights Beacon optofluidic device. Antibody binding to antigen was detected with a fluorescent anti-human IgG secondary Ab (black). b. Left : Schematic of fluorescing beads in the channel above a pen containing an individual B cell indicates antigen-specific reactivity. Top right : False-color still image of positive wells with B cells secreting S2P ecto -reactive antibodies. Reactive antibody diffusing out of a pen is visualized as a plume of fluorescence. Bottom right : False-color still image of positive wells with B cells secreting RBD-mFc-reactive antibodies. c . Representative images of RBD-mFc reactive clones.

    Techniques Used: Functional Assay, Binding Assay, Fluorescence, Clone Assay

    2) Product Images from "Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins"

    Article Title: Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.02284-20

    Structure-based B-cell epitope predictions of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E). (A, C, and E) The predicted B-cell epitopes of SARS-CoV, SARS-CoV-2, and HCoV-229E are shown. The linear (red cartoon) and conformational (yellow sphere) B-cell epitopes were predicted using Bepipred 2.0 or DiscoTope 2.0 and labeled into the corresponding structure via PyMOL. (B, D, and F) The complex structures of the RBDs of SARS-CoV (PDB ID: 2AJF ), SARS-CoV-2 (PDB ID: 6M0J ), and HCoV-229E (PDB ID: 6ATK ) with the receptors (hACE2 and hAPN) are shown. The interface area and the surface area were calculated via PDBePISA. The RBM region of the RBD and the receptors (hACE2 and hAPN) are shown in red and cyan, respectively.
    Figure Legend Snippet: Structure-based B-cell epitope predictions of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E). (A, C, and E) The predicted B-cell epitopes of SARS-CoV, SARS-CoV-2, and HCoV-229E are shown. The linear (red cartoon) and conformational (yellow sphere) B-cell epitopes were predicted using Bepipred 2.0 or DiscoTope 2.0 and labeled into the corresponding structure via PyMOL. (B, D, and F) The complex structures of the RBDs of SARS-CoV (PDB ID: 2AJF ), SARS-CoV-2 (PDB ID: 6M0J ), and HCoV-229E (PDB ID: 6ATK ) with the receptors (hACE2 and hAPN) are shown. The interface area and the surface area were calculated via PDBePISA. The RBM region of the RBD and the receptors (hACE2 and hAPN) are shown in red and cyan, respectively.

    Techniques Used: Labeling

    Immunological analysis of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E) spike proteins. (A and B) Cross-reactivity of the SARS-CoV S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV S-trimer (red) and SARS-CoV RBD (blue) were 2-fold serially diluted and reacted with the S-trimer (A) or RBD (B), respectively. (C and D) Cross-reactivity of the SARS-CoV-2 S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV-2 S-trimer (magenta) and SARS-CoV-2 RBD (cyan) were 10-fold diluted and reacted with SARS-CoV-2 S-trimer (C) and RBD (D). (E and F) Cross-reactivity of the HCoV-229E S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for HCoV-229E S-trimer (orange) and HCoV-229E RBD (green) were 2-fold diluted and reacted with HCoV-229E S-trimer (E) and RBD (F). (G and H) The antibody titers of sera from mice immunized with the HCoV-229E RBD (10 μg, brown; 50 μg, purple). Mouse sera were reacted with the HCoV-229E RBD (G) or S-trimer (H). Mouse sera were serially diluted from a 500-fold dilution. All the data are presented as the mean OD 450 ± SD ( n = 3), and the IgG antibody titers of each serum were calculated as the maximum endpoint dilution that remained positive. (I, J, and K) The neutralization assay of mouse sera from the S-trimer and RBD against SARS-CoV, SARS-CoV-2, and HCoV-229E pseudoviruses. The limit of detection for the assay depends on the initial dilution and is represented by dotted lines; a reciprocal IC 90 titer of 40 was assigned. The data are presented as the mean reciprocal IC 90 titer ± SD ( n = 3). All differences between means with P
    Figure Legend Snippet: Immunological analysis of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E) spike proteins. (A and B) Cross-reactivity of the SARS-CoV S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV S-trimer (red) and SARS-CoV RBD (blue) were 2-fold serially diluted and reacted with the S-trimer (A) or RBD (B), respectively. (C and D) Cross-reactivity of the SARS-CoV-2 S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV-2 S-trimer (magenta) and SARS-CoV-2 RBD (cyan) were 10-fold diluted and reacted with SARS-CoV-2 S-trimer (C) and RBD (D). (E and F) Cross-reactivity of the HCoV-229E S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for HCoV-229E S-trimer (orange) and HCoV-229E RBD (green) were 2-fold diluted and reacted with HCoV-229E S-trimer (E) and RBD (F). (G and H) The antibody titers of sera from mice immunized with the HCoV-229E RBD (10 μg, brown; 50 μg, purple). Mouse sera were reacted with the HCoV-229E RBD (G) or S-trimer (H). Mouse sera were serially diluted from a 500-fold dilution. All the data are presented as the mean OD 450 ± SD ( n = 3), and the IgG antibody titers of each serum were calculated as the maximum endpoint dilution that remained positive. (I, J, and K) The neutralization assay of mouse sera from the S-trimer and RBD against SARS-CoV, SARS-CoV-2, and HCoV-229E pseudoviruses. The limit of detection for the assay depends on the initial dilution and is represented by dotted lines; a reciprocal IC 90 titer of 40 was assigned. The data are presented as the mean reciprocal IC 90 titer ± SD ( n = 3). All differences between means with P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Neutralization

    Structural analysis of S1-RBDs from coronavirus S-trimers. (A) Schematic diagram of coronavirus spike protein organization. S1, receptor-binding subunit; S2, membrane fusion subunit; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Overall structure comparison of coronavirus S-trimers. The S-trimer structures of HCoV-229E (PDB ID: 6U7H ), HCoV-NL63 (PDB ID: 5SZS ), PEDV (PDB ID: 6U7K ), FIPV (PDB ID: 6JX7 ), PDCoV (PDB ID: 6BFU ), IBV (PDB ID: 6CV0 ), SARS-CoV (PDB ID: 5X5B ), SARS-CoV-2 (PDB ID: 6VSB ), MERS-CoV (PDB ID: 5X5F ), HCoV-HKU1 (PDB ID: 5I08 ), HCoV-OC43 (PDB ID: 6OHW ), and mouse hepatitis virus (MHV) (PDB ID: 3JCL ) are shown. The S1-RBDs are colored in magenta. The lengths of the coronavirus S-trimers are shown as previously reported.
    Figure Legend Snippet: Structural analysis of S1-RBDs from coronavirus S-trimers. (A) Schematic diagram of coronavirus spike protein organization. S1, receptor-binding subunit; S2, membrane fusion subunit; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Overall structure comparison of coronavirus S-trimers. The S-trimer structures of HCoV-229E (PDB ID: 6U7H ), HCoV-NL63 (PDB ID: 5SZS ), PEDV (PDB ID: 6U7K ), FIPV (PDB ID: 6JX7 ), PDCoV (PDB ID: 6BFU ), IBV (PDB ID: 6CV0 ), SARS-CoV (PDB ID: 5X5B ), SARS-CoV-2 (PDB ID: 6VSB ), MERS-CoV (PDB ID: 5X5F ), HCoV-HKU1 (PDB ID: 5I08 ), HCoV-OC43 (PDB ID: 6OHW ), and mouse hepatitis virus (MHV) (PDB ID: 3JCL ) are shown. The S1-RBDs are colored in magenta. The lengths of the coronavirus S-trimers are shown as previously reported.

    Techniques Used: Binding Assay

    3) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    4) Product Images from "A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes"

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22926-2

    Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).
    Figure Legend Snippet: Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).

    Techniques Used: Binding Assay, Labeling

    Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).
    Figure Legend Snippet: Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).

    Techniques Used: Infection, Quantitative RT-PCR, Histopathology

    The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.
    Figure Legend Snippet: The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.

    Techniques Used: Binding Assay, SPR Assay, Neutralization

    Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.
    Figure Legend Snippet: Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Neutralization, Infection, Staining

    5) Product Images from "Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2"

    Article Title: Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.02.12.430940

    Mammalian display enables antibody screening of selected clonal lineages. (A) HDR templates are synthesized and transfected into PnP-hybridoma cell lines through CRISPR-Cas9 into the endogenous V H locus. (B) After sorting by flow-cytometry for successful integration with Strep-Tactin and anti-human IgG, (C) enriched hybridoma pools (Pool A and B) are sorted for binding to SARS-CoV-2 S1 or S2.
    Figure Legend Snippet: Mammalian display enables antibody screening of selected clonal lineages. (A) HDR templates are synthesized and transfected into PnP-hybridoma cell lines through CRISPR-Cas9 into the endogenous V H locus. (B) After sorting by flow-cytometry for successful integration with Strep-Tactin and anti-human IgG, (C) enriched hybridoma pools (Pool A and B) are sorted for binding to SARS-CoV-2 S1 or S2.

    Techniques Used: Synthesized, Transfection, CRISPR, Flow Cytometry, Binding Assay

    Validation of SARS-CoV-2 reactive antibodies by deep sequencing and flow cytometry. (A) Sorted cells are amplified using Strep-Tag-specific primers at 5’ and 3’ homology primers for Illumina Next-generation sequencing (see Fig. S4B). After MiXCR alignment, enrichment ratios are calculated to determine SARS-CoV-2 binders. Red tiles indicate highly enriched sequences, blue tiles correspond to strongly depleted sequences post FACS (B) RBD staining of S1 enriched candidates, (C) S1 staining of enriched candidates (D) S2 staining of enriched candidates, three example sequences (mAb-77, mAb-4 and mAb-100) shown on the side stained for both SARS-CoV-2 S2-PE and APC. (E) Dot-plot showing the V H /V L gene usage across all verified reactive (S1, S1-RBD, S2) clonal lineages (F) Sequence similarity network of SARS-CoV-2 reactive sequences and their closest neighbors found in the patients repertoires (based on CDRH3 sequences, edges are drawn between sequences with Levenshtein distance ≤ 3).
    Figure Legend Snippet: Validation of SARS-CoV-2 reactive antibodies by deep sequencing and flow cytometry. (A) Sorted cells are amplified using Strep-Tag-specific primers at 5’ and 3’ homology primers for Illumina Next-generation sequencing (see Fig. S4B). After MiXCR alignment, enrichment ratios are calculated to determine SARS-CoV-2 binders. Red tiles indicate highly enriched sequences, blue tiles correspond to strongly depleted sequences post FACS (B) RBD staining of S1 enriched candidates, (C) S1 staining of enriched candidates (D) S2 staining of enriched candidates, three example sequences (mAb-77, mAb-4 and mAb-100) shown on the side stained for both SARS-CoV-2 S2-PE and APC. (E) Dot-plot showing the V H /V L gene usage across all verified reactive (S1, S1-RBD, S2) clonal lineages (F) Sequence similarity network of SARS-CoV-2 reactive sequences and their closest neighbors found in the patients repertoires (based on CDRH3 sequences, edges are drawn between sequences with Levenshtein distance ≤ 3).

    Techniques Used: Sequencing, Flow Cytometry, Amplification, Strep-tag, Next-Generation Sequencing, FACS, Staining

    Characterization of RBD-specific antibodies for affinity, epitope binning and neutralization. (A-C) Antibodies were purified from supernatant and assayed at 5 different concentrations ranging from approximately 2 to 500 nM [shown here: 125 nM (green), 37.5 nM (yellow), 7.8 nM (purple), 1.9 nM (teal)]. Software calculated fits are shown in red. Binding kinetics (apparent affinity constant kD) to SARS-CoV-2 S1 for FACS-confirmed RBD binders, determined by bio-layer interferometry. (D) Binders were subsequently assayed to perform epitope binning by immobilizing SARS-CoV-2 S1 on the sensors, followed by a first antibody at 166 nM, followed in tandem with a secondary antibody at 78 nM. (E) Additional binding by the secondary molecule indicates an unoccupied epitope (non-competitor, green), while no binding indicates epitope blocking (competitor, red). Self-blocking confirmation can be found on the diagonal. (F) Inhibition of infection of HEK293T-ACE2 cells with SARS-CoV-2 pseudotyped lentivirus.
    Figure Legend Snippet: Characterization of RBD-specific antibodies for affinity, epitope binning and neutralization. (A-C) Antibodies were purified from supernatant and assayed at 5 different concentrations ranging from approximately 2 to 500 nM [shown here: 125 nM (green), 37.5 nM (yellow), 7.8 nM (purple), 1.9 nM (teal)]. Software calculated fits are shown in red. Binding kinetics (apparent affinity constant kD) to SARS-CoV-2 S1 for FACS-confirmed RBD binders, determined by bio-layer interferometry. (D) Binders were subsequently assayed to perform epitope binning by immobilizing SARS-CoV-2 S1 on the sensors, followed by a first antibody at 166 nM, followed in tandem with a secondary antibody at 78 nM. (E) Additional binding by the secondary molecule indicates an unoccupied epitope (non-competitor, green), while no binding indicates epitope blocking (competitor, red). Self-blocking confirmation can be found on the diagonal. (F) Inhibition of infection of HEK293T-ACE2 cells with SARS-CoV-2 pseudotyped lentivirus.

    Techniques Used: Neutralization, Purification, Software, Binding Assay, FACS, Blocking Assay, Inhibition, Infection

    An integrated workflow for interrogating the antibody specificity of PCs from COVID-19 patients. Serum and peripheral blood mononuclear cells (PBMCs) are collected from convalescent COVID-19 patients (with confirmed PCR positive test). Serum is assayed with IgA and IgG ELISAs as well as POCTs. From a subset of 16 patients PCs are isolated from PBMCs by magnetic cell sorting and to then undergo gel encapsulation and barcoding for single-cell sequencing of their antibody heavy and light chain transcripts. Antibody repertoire analysis is performed to identify expanded plasma cell clonal lineages, which are then reformatted into single ORF full-length synthetic antibody genes including homology arms, to allow for single step cloning-free genome editing. The resulting mammalian display library then undergoes high-throughput screening for SARS-CoV-2 binding by flow cytometry and deep sequencing to recover the identity of the corresponding clonal lineages. Supernatant is used to determine cross-reactivity of antibodies within the library with coronavirus antigens.
    Figure Legend Snippet: An integrated workflow for interrogating the antibody specificity of PCs from COVID-19 patients. Serum and peripheral blood mononuclear cells (PBMCs) are collected from convalescent COVID-19 patients (with confirmed PCR positive test). Serum is assayed with IgA and IgG ELISAs as well as POCTs. From a subset of 16 patients PCs are isolated from PBMCs by magnetic cell sorting and to then undergo gel encapsulation and barcoding for single-cell sequencing of their antibody heavy and light chain transcripts. Antibody repertoire analysis is performed to identify expanded plasma cell clonal lineages, which are then reformatted into single ORF full-length synthetic antibody genes including homology arms, to allow for single step cloning-free genome editing. The resulting mammalian display library then undergoes high-throughput screening for SARS-CoV-2 binding by flow cytometry and deep sequencing to recover the identity of the corresponding clonal lineages. Supernatant is used to determine cross-reactivity of antibodies within the library with coronavirus antigens.

    Techniques Used: Polymerase Chain Reaction, Isolation, FACS, Sequencing, Clone Assay, High Throughput Screening Assay, Binding Assay, Flow Cytometry

    6) Product Images from "Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells"

    Article Title: Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1815589

    Validation of the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay. (A) Specificity of the pseudovirus assay. A negative sample panel including 59 human sera and 58 mouse sera were used to determine the specificity of this assay. (B) Reproducibility of the pseudovirus assay. One COVID-19 convalescent patient serum sample was tested 14 times on individual plates in three independent experiments. The virus titer of VSVdG-SARS-CoV-2-Sdel18 pseudovirus was consistent in these assays (MOI=0.05). (C) The correlation of neutralizing titer measured by the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay (ID50, log10) and the wild type SARS-CoV-2 neutralization assay (ID100, log10).
    Figure Legend Snippet: Validation of the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay. (A) Specificity of the pseudovirus assay. A negative sample panel including 59 human sera and 58 mouse sera were used to determine the specificity of this assay. (B) Reproducibility of the pseudovirus assay. One COVID-19 convalescent patient serum sample was tested 14 times on individual plates in three independent experiments. The virus titer of VSVdG-SARS-CoV-2-Sdel18 pseudovirus was consistent in these assays (MOI=0.05). (C) The correlation of neutralizing titer measured by the VSVdG-SARS-CoV-2-Sdel18 pseudovirus assay (ID50, log10) and the wild type SARS-CoV-2 neutralization assay (ID100, log10).

    Techniques Used: Neutralization

    7) Product Images from "A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells"

    Article Title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

    Journal: bioRxiv

    doi: 10.1101/2021.03.31.437907

    Structure of SARS-CoV-2 and differences between B.1 and B.1.1.7 in the Spike protein. (a) SARS-CoV-2 structure. (b) Scheme of S protein. S is composed by the S1 and S2 subunits, which are further subdivided into SP: short peptide, NTD: N-terminal domain, RBD: receptor binding domain, RBM: receptor Binding Motif, FP: fusion peptide, HR1-2: repetitive heptapeptides, TM: transmembrane domain, CP: cytoplasmic peptide. Beside the common D614G mutation, the two most relevant mutations of B.1.1.7 with respect to B.1 are reported: N501Y in RBM and P681H at the S1/S2 cleavage site (indicated by the dashed line).
    Figure Legend Snippet: Structure of SARS-CoV-2 and differences between B.1 and B.1.1.7 in the Spike protein. (a) SARS-CoV-2 structure. (b) Scheme of S protein. S is composed by the S1 and S2 subunits, which are further subdivided into SP: short peptide, NTD: N-terminal domain, RBD: receptor binding domain, RBM: receptor Binding Motif, FP: fusion peptide, HR1-2: repetitive heptapeptides, TM: transmembrane domain, CP: cytoplasmic peptide. Beside the common D614G mutation, the two most relevant mutations of B.1.1.7 with respect to B.1 are reported: N501Y in RBM and P681H at the S1/S2 cleavage site (indicated by the dashed line).

    Techniques Used: Binding Assay, Mutagenesis

    Imaging of cell entry of B.1 and B.1.1.7 SARS-CoV-2. Confocal microscopy images of VeroE6 cells at 1 and 6 hpi; Green: S protein, scale bar: 10 μm.
    Figure Legend Snippet: Imaging of cell entry of B.1 and B.1.1.7 SARS-CoV-2. Confocal microscopy images of VeroE6 cells at 1 and 6 hpi; Green: S protein, scale bar: 10 μm.

    Techniques Used: Imaging, Confocal Microscopy

    Kinetics of cell entry of B.1 and B.1.1.7 SARS-CoV-2. (a) Growth curves showing the release of viral genome into the medium of Vero E6 cells in permanent contact with viruses. (b) Plaque assay on B.1 and B.1.1.7: representative transmission micrographs of plaques are reported; scale bar: 250 μm. (c) Same as (a) but Vero E6 cells were incubated for only 1h with viruses. In (a,c) the viral concentration is expressed by the difference between the cycle threshold (Ct) at the observation time and the Ct at time zero (Ct0). (d) The proportion of cleaved S protein of B.1 and B.1.1.7 at 48 hpi was quantified by densitometry on Western Blot (control: not infected cells).
    Figure Legend Snippet: Kinetics of cell entry of B.1 and B.1.1.7 SARS-CoV-2. (a) Growth curves showing the release of viral genome into the medium of Vero E6 cells in permanent contact with viruses. (b) Plaque assay on B.1 and B.1.1.7: representative transmission micrographs of plaques are reported; scale bar: 250 μm. (c) Same as (a) but Vero E6 cells were incubated for only 1h with viruses. In (a,c) the viral concentration is expressed by the difference between the cycle threshold (Ct) at the observation time and the Ct at time zero (Ct0). (d) The proportion of cleaved S protein of B.1 and B.1.1.7 at 48 hpi was quantified by densitometry on Western Blot (control: not infected cells).

    Techniques Used: Plaque Assay, Transmission Assay, Incubation, Concentration Assay, Western Blot, Infection

    ACE2 and its molecular partners onto the membrane of VeroE6. (a) Membrane distribution of ACE2. (b) Airyscan colocalization image of ACE2 and SARS-CoV-2 RBD. (c) Airyscan colocalization image of ACE2 and Caveolin-1. (d) Airyscan colocalization image of ACE2 (red) and CD71 (green). Red: ACE2, green: RBD, Caveolin-1, CD71. Scale bar: 10 μm (a), 2 μm (b-d).
    Figure Legend Snippet: ACE2 and its molecular partners onto the membrane of VeroE6. (a) Membrane distribution of ACE2. (b) Airyscan colocalization image of ACE2 and SARS-CoV-2 RBD. (c) Airyscan colocalization image of ACE2 and Caveolin-1. (d) Airyscan colocalization image of ACE2 (red) and CD71 (green). Red: ACE2, green: RBD, Caveolin-1, CD71. Scale bar: 10 μm (a), 2 μm (b-d).

    Techniques Used:

    8) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    9) Product Images from "Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding"

    Article Title: Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding

    Journal: Phytomedicine

    doi: 10.1016/j.phymed.2021.153591

    Computational docking prediction of corilagin. A) Molecular docking result showed the best binding pose of interaction, and B) the residues involved and types of interaction of corilagin with the receptor binding domain of the SARS-CoV-2 spike protein. C) Root mean square deviation (RMSD) plot of the SARS-CoV-2 spike protein receptor binding domain (RBD) backbone alone and in complex with corilagin during the 10ns molecular dynamics simulation.
    Figure Legend Snippet: Computational docking prediction of corilagin. A) Molecular docking result showed the best binding pose of interaction, and B) the residues involved and types of interaction of corilagin with the receptor binding domain of the SARS-CoV-2 spike protein. C) Root mean square deviation (RMSD) plot of the SARS-CoV-2 spike protein receptor binding domain (RBD) backbone alone and in complex with corilagin during the 10ns molecular dynamics simulation.

    Techniques Used: Binding Assay

    Effect of corilagin on the interaction of SARS-CoV-2 spike RBD peptide and hACE2 receptor. A) The binding kinetics and steady-state analysis of the interaction between SARS-CoV-2 RBD protein and corilagin was monitored by biolayer interferometry (BLI). B) BLI was used to detect the binding association of hACE2 and corilagin at indicated concentrations. Representative results were shown from 3 independent experiments.
    Figure Legend Snippet: Effect of corilagin on the interaction of SARS-CoV-2 spike RBD peptide and hACE2 receptor. A) The binding kinetics and steady-state analysis of the interaction between SARS-CoV-2 RBD protein and corilagin was monitored by biolayer interferometry (BLI). B) BLI was used to detect the binding association of hACE2 and corilagin at indicated concentrations. Representative results were shown from 3 independent experiments.

    Techniques Used: Binding Assay

    Corilagin suppresses the binding of Spike-RBD on ACE2 receptor in HEK293 cells. A) HEK293 cells were transfected with hACE2-EGFP (green). The transfected HEK293 cells were incubated with mFc-tagged SARS-CoV-2-RBD protein with or without corilagin (25–100μM). Mouse IgG Fc TRITC antibody (red) was used to visualize the binding of SARS-CoV-2-RBD proteins on cell surface. All images were captured by confocal microscopy using a Leica SP8 (× 40 oil immersion objective lens). B) Images of Spike-RBD-ACE2 binding intensity were quantified by Image J. Data were expressed as mean ± S.D., n = 3; * P
    Figure Legend Snippet: Corilagin suppresses the binding of Spike-RBD on ACE2 receptor in HEK293 cells. A) HEK293 cells were transfected with hACE2-EGFP (green). The transfected HEK293 cells were incubated with mFc-tagged SARS-CoV-2-RBD protein with or without corilagin (25–100μM). Mouse IgG Fc TRITC antibody (red) was used to visualize the binding of SARS-CoV-2-RBD proteins on cell surface. All images were captured by confocal microscopy using a Leica SP8 (× 40 oil immersion objective lens). B) Images of Spike-RBD-ACE2 binding intensity were quantified by Image J. Data were expressed as mean ± S.D., n = 3; * P

    Techniques Used: Binding Assay, Transfection, Incubation, Confocal Microscopy

    Corilagin blocks the binding of Spike-RBD peptide on hACE2 receptor. ELISA assay was used to examine the interference of corilagin in its binding to the SARS-CoV-2 RBD protein and hACE2 receptor. Data were expressed as mean ± S.D., representative results were evaluated from 3 independent experiments; * P
    Figure Legend Snippet: Corilagin blocks the binding of Spike-RBD peptide on hACE2 receptor. ELISA assay was used to examine the interference of corilagin in its binding to the SARS-CoV-2 RBD protein and hACE2 receptor. Data were expressed as mean ± S.D., representative results were evaluated from 3 independent experiments; * P

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    10) Product Images from "Infection and transmission of SARS-CoV-2 in London care homes reporting no cases or outbreaks of COVID-19: Prospective observational cohort study, England 2020"

    Article Title: Infection and transmission of SARS-CoV-2 in London care homes reporting no cases or outbreaks of COVID-19: Prospective observational cohort study, England 2020

    Journal: The Lancet Regional Health. Europe

    doi: 10.1016/j.lanepe.2021.100038

    Maximum likelihood phylogeny of 21 SARS-CoV-2 genomes from individuals across the six non-outbreak care homes with PCR positive individuals. Coloured shapes are used to indicate the care home, with circles denoting residents and triangles staff. The phylogenetic tree was rooted using the midpoint of the phylogeny.
    Figure Legend Snippet: Maximum likelihood phylogeny of 21 SARS-CoV-2 genomes from individuals across the six non-outbreak care homes with PCR positive individuals. Coloured shapes are used to indicate the care home, with circles denoting residents and triangles staff. The phylogenetic tree was rooted using the midpoint of the phylogeny.

    Techniques Used: Polymerase Chain Reaction

    a) Summary data showing percentage IgG seropositivity against SARS-CoV-2 nucleocapsid (N) protein (Abbott®) and 95% confidence intervals for care home staff (left panel) and residents (right panel) for each care home (A-M). Dashed line indicates coincident estimated community seroprevalence in London [ 11 ]. b) Correlation of resident and staff seropositivity for each of the 13 care homes (Spearman rank correlation coefficient 0.84, p
    Figure Legend Snippet: a) Summary data showing percentage IgG seropositivity against SARS-CoV-2 nucleocapsid (N) protein (Abbott®) and 95% confidence intervals for care home staff (left panel) and residents (right panel) for each care home (A-M). Dashed line indicates coincident estimated community seroprevalence in London [ 11 ]. b) Correlation of resident and staff seropositivity for each of the 13 care homes (Spearman rank correlation coefficient 0.84, p

    Techniques Used:

    Schematic of care home acute respiratory outbreaks (ARI; blue bars) and total COVID-19 related deaths in care homes in England (pink line) and London (yellow line) reported by ISO week of 2020. The enhanced outbreak testing periods for nasal swabbing SARS-CoV-2 RT-PCR - time-point 0 (T0) - and serology for SARS-CoV-2 antibodies – time-point 1 (T1) - are indicated for the care homes with outbreaks (phase 1 - red), single cases (phase 2 - amber) and those with no reported cases (phase 3 - green).
    Figure Legend Snippet: Schematic of care home acute respiratory outbreaks (ARI; blue bars) and total COVID-19 related deaths in care homes in England (pink line) and London (yellow line) reported by ISO week of 2020. The enhanced outbreak testing periods for nasal swabbing SARS-CoV-2 RT-PCR - time-point 0 (T0) - and serology for SARS-CoV-2 antibodies – time-point 1 (T1) - are indicated for the care homes with outbreaks (phase 1 - red), single cases (phase 2 - amber) and those with no reported cases (phase 3 - green).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    a) Summary data showing IgG serostatus to SARS-CoV-2 N protein by age for non-outbreak care homes (left panel, N=651) and outbreak care homes (right panel, N=586). Statistical analysis using Kruskal-Wallis test non outbreak homes p=0.84. Statistical analysis using Kruskal-Wallis test outbreak homes p=0.58. b) Summary data showing SARS-CoV-2 recombinant N IgG index value (Abbott®) for all seropositive individuals (n=586) for all care homes. Kruskal Wallis test p=0.30. Age bracket of
    Figure Legend Snippet: a) Summary data showing IgG serostatus to SARS-CoV-2 N protein by age for non-outbreak care homes (left panel, N=651) and outbreak care homes (right panel, N=586). Statistical analysis using Kruskal-Wallis test non outbreak homes p=0.84. Statistical analysis using Kruskal-Wallis test outbreak homes p=0.58. b) Summary data showing SARS-CoV-2 recombinant N IgG index value (Abbott®) for all seropositive individuals (n=586) for all care homes. Kruskal Wallis test p=0.30. Age bracket of

    Techniques Used: Recombinant

    11) Product Images from "Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library"

    Article Title: Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library

    Journal: bioRxiv

    doi: 10.1101/2020.05.19.104281

    FACS examination of RBD-binding antibodies to Covid-19 membrane spike on ID8 cells. Soluble scFv-huFc of 22 hits that were RBD ELISA-positive were incubated with equal amount of ID8 cells, followed by goat anti-human Fc (PE conjugated). Stained cells were analyzed to draw dot-plot by FACS. P2 Gating was set based on the background of secondary antibody staining and considered positive. Only clones having positive percentages were shown. Individual clone was labeled in each plot.
    Figure Legend Snippet: FACS examination of RBD-binding antibodies to Covid-19 membrane spike on ID8 cells. Soluble scFv-huFc of 22 hits that were RBD ELISA-positive were incubated with equal amount of ID8 cells, followed by goat anti-human Fc (PE conjugated). Stained cells were analyzed to draw dot-plot by FACS. P2 Gating was set based on the background of secondary antibody staining and considered positive. Only clones having positive percentages were shown. Individual clone was labeled in each plot.

    Techniques Used: FACS, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Clone Assay, Labeling

    MFI change of ACE2 binding to ID8 cells in the presence of serial diluted RBD-antibodies. 8 Covid-19 spike positive antibody binders (1B1, 1B11, 5C2, 1A12, 1A5, 1G6, 1C10 and 1H2) and one isotype negative control antibody (iso-ctrl in the figure) were mixed with ID8 cells at serial diluted concentrations (10 μg/ml down to 0.31 μg/ml, 2 times dilution) before adding of human ACE2-mFc, which has strong interaction with ID8. MFIs (shown in vertical axis) of individual antibody/concentrations cell group (horizonal axis) were complied.
    Figure Legend Snippet: MFI change of ACE2 binding to ID8 cells in the presence of serial diluted RBD-antibodies. 8 Covid-19 spike positive antibody binders (1B1, 1B11, 5C2, 1A12, 1A5, 1G6, 1C10 and 1H2) and one isotype negative control antibody (iso-ctrl in the figure) were mixed with ID8 cells at serial diluted concentrations (10 μg/ml down to 0.31 μg/ml, 2 times dilution) before adding of human ACE2-mFc, which has strong interaction with ID8. MFIs (shown in vertical axis) of individual antibody/concentrations cell group (horizonal axis) were complied.

    Techniques Used: Binding Assay, Negative Control

    BLITZ epitope mapping of Covid-19 RBD-hits antibodies (A). Rotational mutual interaction examination of the six hits (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2) was captured by protein A sensors, followed by RBD binding and one of the six antibodies plus ACE2. RBD was bound well by individual antibodies but not concurrent binding. (B). 1B1 concurrently bound to RBD with any one of the six other antibodies (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2). (C). 5C2 concurrently bound to RBD with any one of the six other antibodies (1B11, 1A5, 1G6, 1C10, A12 and 1H2). (D). 1B1 and 5C2 mutually competed in binding to RBD
    Figure Legend Snippet: BLITZ epitope mapping of Covid-19 RBD-hits antibodies (A). Rotational mutual interaction examination of the six hits (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2) was captured by protein A sensors, followed by RBD binding and one of the six antibodies plus ACE2. RBD was bound well by individual antibodies but not concurrent binding. (B). 1B1 concurrently bound to RBD with any one of the six other antibodies (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2). (C). 5C2 concurrently bound to RBD with any one of the six other antibodies (1B11, 1A5, 1G6, 1C10, A12 and 1H2). (D). 1B1 and 5C2 mutually competed in binding to RBD

    Techniques Used: Binding Assay

    Venn map of Covid-19 hits and ACE2 on RBD Epitope locations on RBD by the isolated 8 hit antibodies were deduced from the above Blitz results and largely categorized into 3 groups (I, II and III). 1B1 and 5C2 have large overlapped sites in RBD and may block the most part of RBD-ACE2 interaction interface. The group III hits (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2) binds to an independent site from 1B1 or 5C2, however may block a minor side of ACE2-RBD interaction interface.
    Figure Legend Snippet: Venn map of Covid-19 hits and ACE2 on RBD Epitope locations on RBD by the isolated 8 hit antibodies were deduced from the above Blitz results and largely categorized into 3 groups (I, II and III). 1B1 and 5C2 have large overlapped sites in RBD and may block the most part of RBD-ACE2 interaction interface. The group III hits (1B11, 1A5, 1G6, 1C10, 1A12 and 1H2) binds to an independent site from 1B1 or 5C2, however may block a minor side of ACE2-RBD interaction interface.

    Techniques Used: Isolation, Blocking Assay

    Validation of Covid-19 spike expression cell line A mammalian cell line ID8 was generated by transfecting 293FT with a pseudo virus that contains full-length Covid-19 S protein, a transmem-brane motif (TM) and a C-terminal 3xFLAG tag. A . The cartoon illustration of the ID8 cells and membrane anchoring of Covid-19 spike (green), C-terminal FLAG (yellow) and binding of ACE2-mFc (blue) to S trimer. B . FACS plot of intracellular staining of FLAG with anti-FLAG-FITC. C . FACS plot of ID8 cell membrane sequential staining of ACE2-mFc, anti-mouse FITC.
    Figure Legend Snippet: Validation of Covid-19 spike expression cell line A mammalian cell line ID8 was generated by transfecting 293FT with a pseudo virus that contains full-length Covid-19 S protein, a transmem-brane motif (TM) and a C-terminal 3xFLAG tag. A . The cartoon illustration of the ID8 cells and membrane anchoring of Covid-19 spike (green), C-terminal FLAG (yellow) and binding of ACE2-mFc (blue) to S trimer. B . FACS plot of intracellular staining of FLAG with anti-FLAG-FITC. C . FACS plot of ID8 cell membrane sequential staining of ACE2-mFc, anti-mouse FITC.

    Techniques Used: Expressing, Generated, Binding Assay, FACS, Staining

    Monophage ELISA screening Covid-19 RBD phage antibodies EHL library was panned with Covid-19 for 2 rounds. Single colonies form 2 nd -round output was picked to prepare monoclonal phage antibody solutions, examined by ELISA with RBD, mouse Fc and 293F cells. Only RBD positive hits were resorted to DNA sequencing of scFv genes. A . Chart of absorbance (A450) on RBD plate ELISA. B . Amino acid sequence multi-alignment of unique scFv clones positive for RBD binding.
    Figure Legend Snippet: Monophage ELISA screening Covid-19 RBD phage antibodies EHL library was panned with Covid-19 for 2 rounds. Single colonies form 2 nd -round output was picked to prepare monoclonal phage antibody solutions, examined by ELISA with RBD, mouse Fc and 293F cells. Only RBD positive hits were resorted to DNA sequencing of scFv genes. A . Chart of absorbance (A450) on RBD plate ELISA. B . Amino acid sequence multi-alignment of unique scFv clones positive for RBD binding.

    Techniques Used: Enzyme-linked Immunosorbent Assay, DNA Sequencing, Sequencing, Clone Assay, Binding Assay

    Cross-reaction examination of Covid-19 RBD hits by ELISA Binding of 8 COVid-19 RBD-hits was examined by ELISA to the spike proteins (named as SARS-S, MERS-S and Covid-19-S) for SARS-COV-1, MERS-COV and SARS-COV-2 (Covid-19) and measured by absorbance (A450). All hits demonstrated strong positive to Covid-19-S (yellow bars) and none cross-reacted to the other two spikes (green and blue bars).
    Figure Legend Snippet: Cross-reaction examination of Covid-19 RBD hits by ELISA Binding of 8 COVid-19 RBD-hits was examined by ELISA to the spike proteins (named as SARS-S, MERS-S and Covid-19-S) for SARS-COV-1, MERS-COV and SARS-COV-2 (Covid-19) and measured by absorbance (A450). All hits demonstrated strong positive to Covid-19-S (yellow bars) and none cross-reacted to the other two spikes (green and blue bars).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    Dual binding examination of Covid-19 RBD-hit antibodies All 8 RBD hits separately were incubated together and ACE2-mFc at around equal concentrations before adding to ID8 cells. Both antibody binding and ACE2 binding were detected by corresponding specific secondary antibodies (different fluorescent conjugation). Upper row from left: FITC-conjugated secondary control, 1A5, 1H2, 1C10, 5C2. Lower row from left: ACE2-mFc (alone, positive control), 1A12, 1G6, 1B11 and 1B1
    Figure Legend Snippet: Dual binding examination of Covid-19 RBD-hit antibodies All 8 RBD hits separately were incubated together and ACE2-mFc at around equal concentrations before adding to ID8 cells. Both antibody binding and ACE2 binding were detected by corresponding specific secondary antibodies (different fluorescent conjugation). Upper row from left: FITC-conjugated secondary control, 1A5, 1H2, 1C10, 5C2. Lower row from left: ACE2-mFc (alone, positive control), 1A12, 1G6, 1B11 and 1B1

    Techniques Used: Binding Assay, Incubation, Conjugation Assay, Positive Control

    12) Product Images from "1,2,3,4,6-Pentagalloyl Glucose, a RBD-ACE2 Binding Inhibitor to Prevent SARS-CoV-2 Infection"

    Article Title: 1,2,3,4,6-Pentagalloyl Glucose, a RBD-ACE2 Binding Inhibitor to Prevent SARS-CoV-2 Infection

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.634176

    Effect of PGG on the interaction of Spike-RBD peptide and hACE2 receptor. (A) BLI was used to monitor the binding association of SARS-CoV-2 RBD and hACE2. (B) The binding kinetics and steady-state analysis of the interaction between immobilized RBD and PGG at indicated concentrations. (C) The binding kinetics and steady-state analysis of the interaction between immobilized ACE2 and PGG at indicated concentrations. Representative results were shown from 3 independent experiments.
    Figure Legend Snippet: Effect of PGG on the interaction of Spike-RBD peptide and hACE2 receptor. (A) BLI was used to monitor the binding association of SARS-CoV-2 RBD and hACE2. (B) The binding kinetics and steady-state analysis of the interaction between immobilized RBD and PGG at indicated concentrations. (C) The binding kinetics and steady-state analysis of the interaction between immobilized ACE2 and PGG at indicated concentrations. Representative results were shown from 3 independent experiments.

    Techniques Used: Binding Assay

    PGG suppresses the binding of Spike-RBD on ACE2 receptor in HEK293 cells. (A) HEK293 cells were transiently transfected with hACE2-EGFP (green). After 24 h, the cells were incubated with supernatant containing mFc-tagged SARS-CoV-2-RBD with or without PGG (25–100 μM) for 40 min. The cells were subsequently fixed and detected with mouse IgG Fc TRITC antibody (red). All images were captured by confocal microscopy using a Leica SP8 (×40 oil immersion objective lens). (B) Images of Spike-RBD-ACE2 binding intensity were quantified by ImageJ. Data were expressed as mean ± S.D., n = 3; * p
    Figure Legend Snippet: PGG suppresses the binding of Spike-RBD on ACE2 receptor in HEK293 cells. (A) HEK293 cells were transiently transfected with hACE2-EGFP (green). After 24 h, the cells were incubated with supernatant containing mFc-tagged SARS-CoV-2-RBD with or without PGG (25–100 μM) for 40 min. The cells were subsequently fixed and detected with mouse IgG Fc TRITC antibody (red). All images were captured by confocal microscopy using a Leica SP8 (×40 oil immersion objective lens). (B) Images of Spike-RBD-ACE2 binding intensity were quantified by ImageJ. Data were expressed as mean ± S.D., n = 3; * p

    Techniques Used: Binding Assay, Transfection, Incubation, Confocal Microscopy

    Computational docking prediction of 1,2,3,4,6-Pentagalloyl glucose (PGG). (A) Molecular docking result showing the best binding pose of interaction, and (B) the residues involved and types of interaction of PGG with the receptor binding domain of the SARS-CoV-2 spike protein. (C) Root mean square deviation (RMSD) plot of the SARS-CoV-2 spike protein receptor binding domain (RBD) backbone alone and in complex with PGG during the 10 ns molecular dynamics simulation.
    Figure Legend Snippet: Computational docking prediction of 1,2,3,4,6-Pentagalloyl glucose (PGG). (A) Molecular docking result showing the best binding pose of interaction, and (B) the residues involved and types of interaction of PGG with the receptor binding domain of the SARS-CoV-2 spike protein. (C) Root mean square deviation (RMSD) plot of the SARS-CoV-2 spike protein receptor binding domain (RBD) backbone alone and in complex with PGG during the 10 ns molecular dynamics simulation.

    Techniques Used: Binding Assay

    13) Product Images from "A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes"

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22926-2

    Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).
    Figure Legend Snippet: Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).

    Techniques Used: Binding Assay, Labeling

    Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).
    Figure Legend Snippet: Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).

    Techniques Used: Infection, Quantitative RT-PCR, Histopathology

    The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.
    Figure Legend Snippet: The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.

    Techniques Used: Binding Assay, SPR Assay, Neutralization

    Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.
    Figure Legend Snippet: Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Neutralization, Infection, Staining

    14) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    15) Product Images from "Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19"

    Article Title: Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00301-9

    Neutralizing antibody responses to SARS-CoV-2 in COVID-19 recovered patients. a Scores showing the COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 RBD protein binding to ACE2 protein by ELISA. b Pie charts showing the proportions of patients with high ( > 50, green) or low (
    Figure Legend Snippet: Neutralizing antibody responses to SARS-CoV-2 in COVID-19 recovered patients. a Scores showing the COVID-19 patient serum-mediated inhibition of the SARS-CoV-2 RBD protein binding to ACE2 protein by ELISA. b Pie charts showing the proportions of patients with high ( > 50, green) or low (

    Techniques Used: Inhibition, Protein Binding, Enzyme-linked Immunosorbent Assay

    Subtypes of neutralizing antibodies to SARS-CoV-2 S proteins in COVID-19 recovered patients. a Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by patient sera (no depletion) or S1 antibody-depleted sera (S1-Abs depletion) or S2 antibody-depleted sera (S2-Abs depletion). The dashed line indicates the cutoff value (6.749) determined by the ROC curve analysis. HC healthy control, NC negative control. b , c Pie charts showing the proportions of patients with different neutralizing antibody (NAb) subtype responses in the total 25 patients ( b ), 8 severe patients ( c , left panel), and 17 moderate and mild patients ( c , right panel) of pseudovirus neutralization positive. d Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by “S1-NAbs only” patient sera with RBD antibody depletion (RBD-Abs depletion) or without RBD antibody depletion (no depletion). The dashed line indicates the cutoff value (6.034) determined by the ROC curve analysis. HC healthy control, NC negative control. e Pie chart showing the proportions of “S1-NAbs only” patients with RBD-Nab-dependent or -independent antibody response. Error bars in a , d indicate SEM
    Figure Legend Snippet: Subtypes of neutralizing antibodies to SARS-CoV-2 S proteins in COVID-19 recovered patients. a Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by patient sera (no depletion) or S1 antibody-depleted sera (S1-Abs depletion) or S2 antibody-depleted sera (S2-Abs depletion). The dashed line indicates the cutoff value (6.749) determined by the ROC curve analysis. HC healthy control, NC negative control. b , c Pie charts showing the proportions of patients with different neutralizing antibody (NAb) subtype responses in the total 25 patients ( b ), 8 severe patients ( c , left panel), and 17 moderate and mild patients ( c , right panel) of pseudovirus neutralization positive. d Blocking of luciferase-encoding SARS-CoV-2 typed pseudovirus into ACE2/293T cells by “S1-NAbs only” patient sera with RBD antibody depletion (RBD-Abs depletion) or without RBD antibody depletion (no depletion). The dashed line indicates the cutoff value (6.034) determined by the ROC curve analysis. HC healthy control, NC negative control. e Pie chart showing the proportions of “S1-NAbs only” patients with RBD-Nab-dependent or -independent antibody response. Error bars in a , d indicate SEM

    Techniques Used: Blocking Assay, Luciferase, Negative Control, Neutralization

    Antibody responses to SARS-CoV-2 in COVID-19 recovered patients with different symptom severity. a – c ELISA binding assays of 100-fold diluted COVID-19 patient sera to ELISA plates after coating with SARS-CoV-2 S1 ( a ), RBD ( b ), and S2 ( c ) proteins. The dashed lines in a – c represent the average values of the healthy control groups. * P
    Figure Legend Snippet: Antibody responses to SARS-CoV-2 in COVID-19 recovered patients with different symptom severity. a – c ELISA binding assays of 100-fold diluted COVID-19 patient sera to ELISA plates after coating with SARS-CoV-2 S1 ( a ), RBD ( b ), and S2 ( c ) proteins. The dashed lines in a – c represent the average values of the healthy control groups. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    16) Product Images from "The efficacy assessment of convalescent plasma therapy for COVID-19 patients: a multi-center case series"

    Article Title: The efficacy assessment of convalescent plasma therapy for COVID-19 patients: a multi-center case series

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00329-x

    Changes of laboratory results before and at day 1–5 after convalescent plasma transfusion. a , b SARS-CoV-2 specific IgG and IgM levels, respectively, determined by MCLIA. c , d Cycle threshold (Ct) values of ORF1ab-gene and N-gene, respectively. A Ct value of 40 was defined as undetectable. e PaO 2 /FiO 2 (normal range: 400–500 mmHg). f White blood cell count (normal range: 3.5–9.5). g Lymphocyte count (normal range: 1.1–3.2). h C-reactive protein (normal range:
    Figure Legend Snippet: Changes of laboratory results before and at day 1–5 after convalescent plasma transfusion. a , b SARS-CoV-2 specific IgG and IgM levels, respectively, determined by MCLIA. c , d Cycle threshold (Ct) values of ORF1ab-gene and N-gene, respectively. A Ct value of 40 was defined as undetectable. e PaO 2 /FiO 2 (normal range: 400–500 mmHg). f White blood cell count (normal range: 3.5–9.5). g Lymphocyte count (normal range: 1.1–3.2). h C-reactive protein (normal range:

    Techniques Used: Cell Counting

    SARS-CoV-2 specific antibody levels of CP samples measured by serology tests, receptor-binding assay, and pseudovirus based neutralization assay. a The correlations among anti-SARS-CoV-2 specific IgG and IgM titers detected by commercial MCLIA kits, anti-S-RBD and anti-NP specific IgG titers determined by in-house ELISA assays, inhibition activity measured by a receptor-binding assay, and neutralizing antibody titer measured by a pseudovirus based neutralization assay. b Comparisons of antibody levels between CP samples collected before and after 21 days from symptom onset. MCLIA magnetic chemiluminescence enzyme immunoassay, ELISA enzyme-linked immunosorbent assay, RBD receptor binding domains, NP nucleoprotein, IT50 inhibitory titer which was calculated with the dilution of plasma that inhibits 50% RBD-Fc binding to receptor ACE2, NAT50 neutralizing antibody titer which was calculated with the highest dilution of plasma that resulted in a 50% reduction of virus infection, GMT geometric mean titer, CI confidence interval
    Figure Legend Snippet: SARS-CoV-2 specific antibody levels of CP samples measured by serology tests, receptor-binding assay, and pseudovirus based neutralization assay. a The correlations among anti-SARS-CoV-2 specific IgG and IgM titers detected by commercial MCLIA kits, anti-S-RBD and anti-NP specific IgG titers determined by in-house ELISA assays, inhibition activity measured by a receptor-binding assay, and neutralizing antibody titer measured by a pseudovirus based neutralization assay. b Comparisons of antibody levels between CP samples collected before and after 21 days from symptom onset. MCLIA magnetic chemiluminescence enzyme immunoassay, ELISA enzyme-linked immunosorbent assay, RBD receptor binding domains, NP nucleoprotein, IT50 inhibitory titer which was calculated with the dilution of plasma that inhibits 50% RBD-Fc binding to receptor ACE2, NAT50 neutralizing antibody titer which was calculated with the highest dilution of plasma that resulted in a 50% reduction of virus infection, GMT geometric mean titer, CI confidence interval

    Techniques Used: Reporter Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Inhibition, Activity Assay, Binding Assay, Infection

    17) Product Images from "A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351"

    Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

    Journal: mAbs

    doi: 10.1080/19420862.2021.1930636

    Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software
    Figure Legend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

    Techniques Used: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization, Plaque Reduction Neutralization Test

    Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software
    Figure Legend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

    Techniques Used: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

    Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity
    Figure Legend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

    Techniques Used: Clone Assay, Plasmid Preparation, FACS, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

    Effects of Fc modification on the ADE activity of JMB2002. (a) Binding of Ab2001.08 and JMB2002 to FcγRs was determined by BLI. His-tagged FcγR was loaded onto the HIS1K sensor, and serially diluted antibodies bound to the receptor on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. (b-d) ADE activity was measured using a pseudotyped SARS-CoV-2 system containing a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to FcγR-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. Trastuzumab was used as the irrelevant IgG control
    Figure Legend Snippet: Effects of Fc modification on the ADE activity of JMB2002. (a) Binding of Ab2001.08 and JMB2002 to FcγRs was determined by BLI. His-tagged FcγR was loaded onto the HIS1K sensor, and serially diluted antibodies bound to the receptor on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. (b-d) ADE activity was measured using a pseudotyped SARS-CoV-2 system containing a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to FcγR-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. Trastuzumab was used as the irrelevant IgG control

    Techniques Used: Modification, Activity Assay, Binding Assay, Software, Luciferase, Expressing, Incubation, Infection

    Prophylactic and therapeutic efficacies of JMB2002 against SARS-CoV-2 infection in rhesus macaques. (a) Schematic representation of the design of the in vivo animal experiment. Five monkeys were divided into three groups: the control group (one animal, C1), prophylactic group (two animals, PA1 and PA2), and therapeutic group (two animals, AC1 and AC2). In the prophylactic group, a single dose of 20 mg/kg JMB2002 was intravenously injected into the animals before SARS-CoV-2 infection. The next day, all monkeys were infected with virus (1 × 10 5 TCID 50 ) via intratracheal inoculation. In the therapeutic group, 50 mg/kg JMB2002 was injected at 1 and 3 dpi, whereas in the control group, a single dose of 20 mg/kg irrelevant IgG control was administered at 1 dpi. (b) The viral load in oropharyngeal swabs was monitored for 7 days by qRT-PCR. The dotted line indicates the copy number detection limit. (c) Histopathological and immunohistochemical characterization of lung tissues. All animals were euthanized and necropsied at 7 dpi. The tissue samples were collected, fixed in 10% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or Masson’s trichrome before observation by light microscopy. Scale bar = 100 μm
    Figure Legend Snippet: Prophylactic and therapeutic efficacies of JMB2002 against SARS-CoV-2 infection in rhesus macaques. (a) Schematic representation of the design of the in vivo animal experiment. Five monkeys were divided into three groups: the control group (one animal, C1), prophylactic group (two animals, PA1 and PA2), and therapeutic group (two animals, AC1 and AC2). In the prophylactic group, a single dose of 20 mg/kg JMB2002 was intravenously injected into the animals before SARS-CoV-2 infection. The next day, all monkeys were infected with virus (1 × 10 5 TCID 50 ) via intratracheal inoculation. In the therapeutic group, 50 mg/kg JMB2002 was injected at 1 and 3 dpi, whereas in the control group, a single dose of 20 mg/kg irrelevant IgG control was administered at 1 dpi. (b) The viral load in oropharyngeal swabs was monitored for 7 days by qRT-PCR. The dotted line indicates the copy number detection limit. (c) Histopathological and immunohistochemical characterization of lung tissues. All animals were euthanized and necropsied at 7 dpi. The tissue samples were collected, fixed in 10% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or Masson’s trichrome before observation by light microscopy. Scale bar = 100 μm

    Techniques Used: Infection, In Vivo, Injection, Quantitative RT-PCR, Immunohistochemistry, Staining, Light Microscopy

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    Immunofluorescence:

    Article Title: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells
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    Mouse Assay:

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2
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    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2
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    Binding Assay:

    Article Title: Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2
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    Staining:

    Article Title: Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2
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    Generated:

    Article Title: Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells
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    Sino Biological mouse igg1 fc domain
    Functional assays from single antigen-reactive B cells. a. Schematic of detection of antigen-specific antibody . Biotinylated antigen (dark grey) was coupled to a streptavidin-conjugated polystyrene bead (light grey). Antibodies (blue) are secreted by single B cells loaded into individual NanoPens on the Berkeley Lights Beacon optofluidic device. Antibody binding to antigen was detected with a fluorescent anti-human <t>IgG</t> secondary Ab (black). b. Left : Schematic of fluorescing beads in the channel above a pen containing an individual B cell indicates antigen-specific reactivity. Top right : False-color still image of positive wells with B cells secreting S2P ecto -reactive antibodies. Reactive antibody diffusing out of a pen is visualized as a plume of fluorescence. Bottom right : False-color still image of positive wells with B cells secreting RBD-mFc-reactive antibodies. c . Representative images of RBD-mFc reactive clones.
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    Functional assays from single antigen-reactive B cells. a. Schematic of detection of antigen-specific antibody . Biotinylated antigen (dark grey) was coupled to a streptavidin-conjugated polystyrene bead (light grey). Antibodies (blue) are secreted by single B cells loaded into individual NanoPens on the Berkeley Lights Beacon optofluidic device. Antibody binding to antigen was detected with a fluorescent anti-human IgG secondary Ab (black). b. Left : Schematic of fluorescing beads in the channel above a pen containing an individual B cell indicates antigen-specific reactivity. Top right : False-color still image of positive wells with B cells secreting S2P ecto -reactive antibodies. Reactive antibody diffusing out of a pen is visualized as a plume of fluorescence. Bottom right : False-color still image of positive wells with B cells secreting RBD-mFc-reactive antibodies. c . Representative images of RBD-mFc reactive clones.

    Journal: bioRxiv

    Article Title: Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

    doi: 10.1101/2020.05.12.091462

    Figure Lengend Snippet: Functional assays from single antigen-reactive B cells. a. Schematic of detection of antigen-specific antibody . Biotinylated antigen (dark grey) was coupled to a streptavidin-conjugated polystyrene bead (light grey). Antibodies (blue) are secreted by single B cells loaded into individual NanoPens on the Berkeley Lights Beacon optofluidic device. Antibody binding to antigen was detected with a fluorescent anti-human IgG secondary Ab (black). b. Left : Schematic of fluorescing beads in the channel above a pen containing an individual B cell indicates antigen-specific reactivity. Top right : False-color still image of positive wells with B cells secreting S2P ecto -reactive antibodies. Reactive antibody diffusing out of a pen is visualized as a plume of fluorescence. Bottom right : False-color still image of positive wells with B cells secreting RBD-mFc-reactive antibodies. c . Representative images of RBD-mFc reactive clones.

    Article Snippet: RBD protein fused to mouse IgG1 Fc domain (designated RBD-mFc), was purchased from Sino Biological (40592-V05H).

    Techniques: Functional Assay, Binding Assay, Fluorescence, Clone Assay

    Structure-based B-cell epitope predictions of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E). (A, C, and E) The predicted B-cell epitopes of SARS-CoV, SARS-CoV-2, and HCoV-229E are shown. The linear (red cartoon) and conformational (yellow sphere) B-cell epitopes were predicted using Bepipred 2.0 or DiscoTope 2.0 and labeled into the corresponding structure via PyMOL. (B, D, and F) The complex structures of the RBDs of SARS-CoV (PDB ID: 2AJF ), SARS-CoV-2 (PDB ID: 6M0J ), and HCoV-229E (PDB ID: 6ATK ) with the receptors (hACE2 and hAPN) are shown. The interface area and the surface area were calculated via PDBePISA. The RBM region of the RBD and the receptors (hACE2 and hAPN) are shown in red and cyan, respectively.

    Journal: Journal of Virology

    Article Title: Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

    doi: 10.1128/JVI.02284-20

    Figure Lengend Snippet: Structure-based B-cell epitope predictions of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E). (A, C, and E) The predicted B-cell epitopes of SARS-CoV, SARS-CoV-2, and HCoV-229E are shown. The linear (red cartoon) and conformational (yellow sphere) B-cell epitopes were predicted using Bepipred 2.0 or DiscoTope 2.0 and labeled into the corresponding structure via PyMOL. (B, D, and F) The complex structures of the RBDs of SARS-CoV (PDB ID: 2AJF ), SARS-CoV-2 (PDB ID: 6M0J ), and HCoV-229E (PDB ID: 6ATK ) with the receptors (hACE2 and hAPN) are shown. The interface area and the surface area were calculated via PDBePISA. The RBM region of the RBD and the receptors (hACE2 and hAPN) are shown in red and cyan, respectively.

    Article Snippet: Besides, for SARS-CoV-2, the S-trimer (item no. 40589-V08B1) and S1-RBD (item no. 40592-V05H) were purchased from Sino Biological, Inc.

    Techniques: Labeling

    Immunological analysis of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E) spike proteins. (A and B) Cross-reactivity of the SARS-CoV S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV S-trimer (red) and SARS-CoV RBD (blue) were 2-fold serially diluted and reacted with the S-trimer (A) or RBD (B), respectively. (C and D) Cross-reactivity of the SARS-CoV-2 S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV-2 S-trimer (magenta) and SARS-CoV-2 RBD (cyan) were 10-fold diluted and reacted with SARS-CoV-2 S-trimer (C) and RBD (D). (E and F) Cross-reactivity of the HCoV-229E S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for HCoV-229E S-trimer (orange) and HCoV-229E RBD (green) were 2-fold diluted and reacted with HCoV-229E S-trimer (E) and RBD (F). (G and H) The antibody titers of sera from mice immunized with the HCoV-229E RBD (10 μg, brown; 50 μg, purple). Mouse sera were reacted with the HCoV-229E RBD (G) or S-trimer (H). Mouse sera were serially diluted from a 500-fold dilution. All the data are presented as the mean OD 450 ± SD ( n = 3), and the IgG antibody titers of each serum were calculated as the maximum endpoint dilution that remained positive. (I, J, and K) The neutralization assay of mouse sera from the S-trimer and RBD against SARS-CoV, SARS-CoV-2, and HCoV-229E pseudoviruses. The limit of detection for the assay depends on the initial dilution and is represented by dotted lines; a reciprocal IC 90 titer of 40 was assigned. The data are presented as the mean reciprocal IC 90 titer ± SD ( n = 3). All differences between means with P

    Journal: Journal of Virology

    Article Title: Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

    doi: 10.1128/JVI.02284-20

    Figure Lengend Snippet: Immunological analysis of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E) spike proteins. (A and B) Cross-reactivity of the SARS-CoV S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV S-trimer (red) and SARS-CoV RBD (blue) were 2-fold serially diluted and reacted with the S-trimer (A) or RBD (B), respectively. (C and D) Cross-reactivity of the SARS-CoV-2 S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV-2 S-trimer (magenta) and SARS-CoV-2 RBD (cyan) were 10-fold diluted and reacted with SARS-CoV-2 S-trimer (C) and RBD (D). (E and F) Cross-reactivity of the HCoV-229E S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for HCoV-229E S-trimer (orange) and HCoV-229E RBD (green) were 2-fold diluted and reacted with HCoV-229E S-trimer (E) and RBD (F). (G and H) The antibody titers of sera from mice immunized with the HCoV-229E RBD (10 μg, brown; 50 μg, purple). Mouse sera were reacted with the HCoV-229E RBD (G) or S-trimer (H). Mouse sera were serially diluted from a 500-fold dilution. All the data are presented as the mean OD 450 ± SD ( n = 3), and the IgG antibody titers of each serum were calculated as the maximum endpoint dilution that remained positive. (I, J, and K) The neutralization assay of mouse sera from the S-trimer and RBD against SARS-CoV, SARS-CoV-2, and HCoV-229E pseudoviruses. The limit of detection for the assay depends on the initial dilution and is represented by dotted lines; a reciprocal IC 90 titer of 40 was assigned. The data are presented as the mean reciprocal IC 90 titer ± SD ( n = 3). All differences between means with P

    Article Snippet: Besides, for SARS-CoV-2, the S-trimer (item no. 40589-V08B1) and S1-RBD (item no. 40592-V05H) were purchased from Sino Biological, Inc.

    Techniques: Enzyme-linked Immunosorbent Assay, Mouse Assay, Neutralization

    Structural analysis of S1-RBDs from coronavirus S-trimers. (A) Schematic diagram of coronavirus spike protein organization. S1, receptor-binding subunit; S2, membrane fusion subunit; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Overall structure comparison of coronavirus S-trimers. The S-trimer structures of HCoV-229E (PDB ID: 6U7H ), HCoV-NL63 (PDB ID: 5SZS ), PEDV (PDB ID: 6U7K ), FIPV (PDB ID: 6JX7 ), PDCoV (PDB ID: 6BFU ), IBV (PDB ID: 6CV0 ), SARS-CoV (PDB ID: 5X5B ), SARS-CoV-2 (PDB ID: 6VSB ), MERS-CoV (PDB ID: 5X5F ), HCoV-HKU1 (PDB ID: 5I08 ), HCoV-OC43 (PDB ID: 6OHW ), and mouse hepatitis virus (MHV) (PDB ID: 3JCL ) are shown. The S1-RBDs are colored in magenta. The lengths of the coronavirus S-trimers are shown as previously reported.

    Journal: Journal of Virology

    Article Title: Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

    doi: 10.1128/JVI.02284-20

    Figure Lengend Snippet: Structural analysis of S1-RBDs from coronavirus S-trimers. (A) Schematic diagram of coronavirus spike protein organization. S1, receptor-binding subunit; S2, membrane fusion subunit; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Overall structure comparison of coronavirus S-trimers. The S-trimer structures of HCoV-229E (PDB ID: 6U7H ), HCoV-NL63 (PDB ID: 5SZS ), PEDV (PDB ID: 6U7K ), FIPV (PDB ID: 6JX7 ), PDCoV (PDB ID: 6BFU ), IBV (PDB ID: 6CV0 ), SARS-CoV (PDB ID: 5X5B ), SARS-CoV-2 (PDB ID: 6VSB ), MERS-CoV (PDB ID: 5X5F ), HCoV-HKU1 (PDB ID: 5I08 ), HCoV-OC43 (PDB ID: 6OHW ), and mouse hepatitis virus (MHV) (PDB ID: 3JCL ) are shown. The S1-RBDs are colored in magenta. The lengths of the coronavirus S-trimers are shown as previously reported.

    Article Snippet: Besides, for SARS-CoV-2, the S-trimer (item no. 40589-V08B1) and S1-RBD (item no. 40592-V05H) were purchased from Sino Biological, Inc.

    Techniques: Binding Assay

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Journal: Communications Biology

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    doi: 10.1038/s42003-021-02029-w

    Figure Lengend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Article Snippet: Another two mice (named Q14, Q15) were immunized in 10-day intervals with mixtures of Spike S1 protein (Cat 40591-V02H, Sino Biological) and RBD protein (Cat 40592-V05H, Sino Biological) and boosted by 70 μg mixtures (35 μg for each protein) after three rounds immunization.

    Techniques: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Journal: Communications Biology

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    doi: 10.1038/s42003-021-02029-w

    Figure Lengend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Article Snippet: Another two mice (named Q14, Q15) were immunized in 10-day intervals with mixtures of Spike S1 protein (Cat 40591-V02H, Sino Biological) and RBD protein (Cat 40592-V05H, Sino Biological) and boosted by 70 μg mixtures (35 μg for each protein) after three rounds immunization.

    Techniques: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Journal: Communications Biology

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    doi: 10.1038/s42003-021-02029-w

    Figure Lengend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Article Snippet: Another two mice (named Q14, Q15) were immunized in 10-day intervals with mixtures of Spike S1 protein (Cat 40591-V02H, Sino Biological) and RBD protein (Cat 40592-V05H, Sino Biological) and boosted by 70 μg mixtures (35 μg for each protein) after three rounds immunization.

    Techniques: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    doi: 10.1038/s41467-021-22926-2

    Figure Lengend Snippet: Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).

    Article Snippet: The 384-well plate (Corning, Catalog #3700) was coated overnight at 4 °C with PBS containing 30 μL 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein (Sino Biological, Catalog #40589-V08B1), or SARS-CoV-2 Spike RBD-mFc recombinant protein (Sino Biological, Catalog #40592-V05H) or S1-mFc (Sino Biological, Catalog #40591-V05H1).

    Techniques: Binding Assay, Labeling

    Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    doi: 10.1038/s41467-021-22926-2

    Figure Lengend Snippet: Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).

    Article Snippet: The 384-well plate (Corning, Catalog #3700) was coated overnight at 4 °C with PBS containing 30 μL 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein (Sino Biological, Catalog #40589-V08B1), or SARS-CoV-2 Spike RBD-mFc recombinant protein (Sino Biological, Catalog #40592-V05H) or S1-mFc (Sino Biological, Catalog #40591-V05H1).

    Techniques: Infection, Quantitative RT-PCR, Histopathology

    The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    doi: 10.1038/s41467-021-22926-2

    Figure Lengend Snippet: The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.

    Article Snippet: The 384-well plate (Corning, Catalog #3700) was coated overnight at 4 °C with PBS containing 30 μL 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein (Sino Biological, Catalog #40589-V08B1), or SARS-CoV-2 Spike RBD-mFc recombinant protein (Sino Biological, Catalog #40592-V05H) or S1-mFc (Sino Biological, Catalog #40591-V05H1).

    Techniques: Binding Assay, SPR Assay, Neutralization

    Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.

    Journal: Nature Communications

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    doi: 10.1038/s41467-021-22926-2

    Figure Lengend Snippet: Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.

    Article Snippet: The 384-well plate (Corning, Catalog #3700) was coated overnight at 4 °C with PBS containing 30 μL 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein (Sino Biological, Catalog #40589-V08B1), or SARS-CoV-2 Spike RBD-mFc recombinant protein (Sino Biological, Catalog #40592-V05H) or S1-mFc (Sino Biological, Catalog #40591-V05H1).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Neutralization, Infection, Staining