s1 protein  (Sino Biological)


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    Name:
    SARS CoV Spike S1 Protein S1 Subunit His Tag
    Description:
    A DNA sequence encoding the S1 subunit of SARS CoV isolate WH20 spike AAX16192 1 Met1 Arg667 was expressed with a C terminal polyhistidine tag
    Catalog Number:
    40150-V08B1
    Price:
    None
    Category:
    recombinant protein
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological s1 protein
    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the <t>S1</t> protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    A DNA sequence encoding the S1 subunit of SARS CoV isolate WH20 spike AAX16192 1 Met1 Arg667 was expressed with a C terminal polyhistidine tag
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    Images

    1) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    2) Product Images from "Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples"

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

    Antibody affinity screening. (A) Illustration of the assay mechanism, which uses a single-step ELISA. The sample-to-answer time of this assay is 8 minutes. (B)-(C) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2 (B) and SARS-CoV (C). The solid lines are the linear fit of the data in the log-log scale.
    Figure Legend Snippet: Antibody affinity screening. (A) Illustration of the assay mechanism, which uses a single-step ELISA. The sample-to-answer time of this assay is 8 minutes. (B)-(C) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2 (B) and SARS-CoV (C). The solid lines are the linear fit of the data in the log-log scale.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    S1 protein detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 20 minutes. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3×standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein and SARS-CoV S1 is 0.4 ng/mL and 0.2 ng/mL, respectively. (C) Calibration curves for S1 proteins between 0.78 and 200 ng/mL. The error bars are generated from duplicate measurements.
    Figure Legend Snippet: S1 protein detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 20 minutes. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3×standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein and SARS-CoV S1 is 0.4 ng/mL and 0.2 ng/mL, respectively. (C) Calibration curves for S1 proteins between 0.78 and 200 ng/mL. The error bars are generated from duplicate measurements.

    Techniques Used: Generated

    Detection of anti-S1 IgG. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 15 minutes. (B)-(D) Detection of S1 specific IgG in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (B), D001 in (C), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. See also Figure S3 for the entire dynamic range of CR3022, D001, and D006, and their respective lower limits of detection.
    Figure Legend Snippet: Detection of anti-S1 IgG. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 15 minutes. (B)-(D) Detection of S1 specific IgG in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (B), D001 in (C), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. See also Figure S3 for the entire dynamic range of CR3022, D001, and D006, and their respective lower limits of detection.

    Techniques Used: Generated

    3) Product Images from "Structural and functional analysis of a potent sarbecovirus neutralizing antibody"

    Article Title: Structural and functional analysis of a potent sarbecovirus neutralizing antibody

    Journal: bioRxiv

    doi: 10.1101/2020.04.07.023903

    Mechanism of neutralization of S309 mAb. a-b , Ribbon diagrams of S309 and ACE2 bound to SARS-CoV-2 S B . This composite model was generated using the SARS-CoV-2 S/S309 cryoEM structure reported here and a crystal structure of SARS-CoV-2 S bound to ACE2 16 . c , Competition of S309 or S230 mAbs with ACE2 to bind to SARS-CoV S B (left panel) and SARS-CoV-2 S B (right panel). ACE2 was immobilized at the surface of biosensors before incubation with S B domain alone or S B precomplexed with mAbs. The vertical dashed line indicates the start of the association of mAb-complexed or free S B to solid-phase ACE2. d , Neutralization of SARS-CoV-MLV by S309 IgG1 or S309 Fab, plotted in nM (means ± SD is shown, one out of two experiments is shown). e , mAb-mediated ADCC using primary NK effector cells and SARS-CoV-2 S-expressing ExpiCHO as target cells. Bar graph shows the average area under the curve (AUC) for the responses of 3-4 donors genotyped for their FcγRIIIa (mean±SD, from two independent experiments). f , Activation of high affinity (V158) or low affinity (F158) FcγRIIIa was measured using Jurkat reporter cells and SARS-CoV-2 S-expressing ExpiCHO as target cells (one experiment, one or two measurements per mAb). g , mAb-mediated ADCP using Cell Trace Violet-labelled PBMCs as phagocytic cells and PKF67-labelled SARS-CoV-2 S-expressing ExpiCHO as target cells. Bar graph shows the average area under the curve (AUC) for the responses of four donor (mean±SD, from two independent experiments). h , Activation of FcγRIIa measured using Jurkat reporter cells and SARS-CoV-2 S-expressing ExpiCHO as target cells (one experiment, one or two measurements per mAb).
    Figure Legend Snippet: Mechanism of neutralization of S309 mAb. a-b , Ribbon diagrams of S309 and ACE2 bound to SARS-CoV-2 S B . This composite model was generated using the SARS-CoV-2 S/S309 cryoEM structure reported here and a crystal structure of SARS-CoV-2 S bound to ACE2 16 . c , Competition of S309 or S230 mAbs with ACE2 to bind to SARS-CoV S B (left panel) and SARS-CoV-2 S B (right panel). ACE2 was immobilized at the surface of biosensors before incubation with S B domain alone or S B precomplexed with mAbs. The vertical dashed line indicates the start of the association of mAb-complexed or free S B to solid-phase ACE2. d , Neutralization of SARS-CoV-MLV by S309 IgG1 or S309 Fab, plotted in nM (means ± SD is shown, one out of two experiments is shown). e , mAb-mediated ADCC using primary NK effector cells and SARS-CoV-2 S-expressing ExpiCHO as target cells. Bar graph shows the average area under the curve (AUC) for the responses of 3-4 donors genotyped for their FcγRIIIa (mean±SD, from two independent experiments). f , Activation of high affinity (V158) or low affinity (F158) FcγRIIIa was measured using Jurkat reporter cells and SARS-CoV-2 S-expressing ExpiCHO as target cells (one experiment, one or two measurements per mAb). g , mAb-mediated ADCP using Cell Trace Violet-labelled PBMCs as phagocytic cells and PKF67-labelled SARS-CoV-2 S-expressing ExpiCHO as target cells. Bar graph shows the average area under the curve (AUC) for the responses of four donor (mean±SD, from two independent experiments). h , Activation of FcγRIIa measured using Jurkat reporter cells and SARS-CoV-2 S-expressing ExpiCHO as target cells (one experiment, one or two measurements per mAb).

    Techniques Used: Neutralization, Generated, Incubation, Expressing, Activation Assay

    MAb cocktails enhance SARS-CoV-2 neutralization. a, Heat map showing the competition of mAb pairs for binding to the SARS-CoV S B domain as measured by biolayer interferometry (as shown in Extended Data Fig. 9). b , Competition of mAb pairs for binding to the SARS-CoV-2 S B domain. c-d , Neutralization of SARS-CoV-2-MLV by S309 combined with an equimolar amount of S304 or S315 mAbs. For mAb cocktails the concentration on the x axis is that of the individual mAbs.
    Figure Legend Snippet: MAb cocktails enhance SARS-CoV-2 neutralization. a, Heat map showing the competition of mAb pairs for binding to the SARS-CoV S B domain as measured by biolayer interferometry (as shown in Extended Data Fig. 9). b , Competition of mAb pairs for binding to the SARS-CoV-2 S B domain. c-d , Neutralization of SARS-CoV-2-MLV by S309 combined with an equimolar amount of S304 or S315 mAbs. For mAb cocktails the concentration on the x axis is that of the individual mAbs.

    Techniques Used: Neutralization, Binding Assay, Concentration Assay

    Identification of a potent SARS-CoV-2 neutralizing mAb from a SARS survivor. a-b , Binding of a panel of mAbs isolated from a SARS-immune patient to the SARS-CoV-2 ( a ) or SARS-CoV ( b ) S glycoproteins expressed at the surface of ExpiCHO cells (symbols are means of duplicates from one experiment). c-d , Affinity measurement of S309 full-length IgG1 and Fab for SARS-CoV-2 and SARS-CoV S B domains measured using biolayer interferometry. e , Neutralization of SARS-CoV-2-MLV, SARS-CoV-MLV (bearing S from various isolates) and other sarbecovirus isolates by mAb S309. f , Neutralization of authentic SARS-CoV-2 (strain n-CoV/USA_WA1/2020) by mAbs as measured by a focus-forming assay on Vero E6 cells. ( e-f ) mean±SD ( e ) or means ( f) of duplicates are shown. One representative out of two experiments is shown.
    Figure Legend Snippet: Identification of a potent SARS-CoV-2 neutralizing mAb from a SARS survivor. a-b , Binding of a panel of mAbs isolated from a SARS-immune patient to the SARS-CoV-2 ( a ) or SARS-CoV ( b ) S glycoproteins expressed at the surface of ExpiCHO cells (symbols are means of duplicates from one experiment). c-d , Affinity measurement of S309 full-length IgG1 and Fab for SARS-CoV-2 and SARS-CoV S B domains measured using biolayer interferometry. e , Neutralization of SARS-CoV-2-MLV, SARS-CoV-MLV (bearing S from various isolates) and other sarbecovirus isolates by mAb S309. f , Neutralization of authentic SARS-CoV-2 (strain n-CoV/USA_WA1/2020) by mAbs as measured by a focus-forming assay on Vero E6 cells. ( e-f ) mean±SD ( e ) or means ( f) of duplicates are shown. One representative out of two experiments is shown.

    Techniques Used: Binding Assay, Isolation, Neutralization, Focus Forming Assay

    CryoEM structures of the SARS-CoV-2 S glycoprotein in complex with the S309 neutralizing antibody Fab fragment. a , Ribbon diagram of the partially open SARS-CoV-2 S trimer (one S B domain is open) bound to three S309 Fabs. b-c , Ribbon diagrams of the closed SARS-CoV-2 S trimer bound to three S309 Fabs shown in two orthogonal orientations. d , Close-up view of the S309 epitope showing the contacts formed with the core fucose (labeled with a star) and the core N-acetyl-glucosamine of the oligosaccharide at position N343. e , Close-up view of the S309 epitope showing the 20-residue long CDRH3 siting atop the S B helix comprising residues 337-344. The oligosaccharide at position N343 is omitted for clarity. In panels ( c-d ), selected residues involved in interactions between S309 and SARS-CoV-2 S are shown. F , Molecular surface representation of the SARS-CoV-2 S trimer showing the S309 footprint colored by residue conservation on one protomer among SARS-CoV-2 and SARS-CoV S glycoproteins. The other two protomers are colored pink and gold.
    Figure Legend Snippet: CryoEM structures of the SARS-CoV-2 S glycoprotein in complex with the S309 neutralizing antibody Fab fragment. a , Ribbon diagram of the partially open SARS-CoV-2 S trimer (one S B domain is open) bound to three S309 Fabs. b-c , Ribbon diagrams of the closed SARS-CoV-2 S trimer bound to three S309 Fabs shown in two orthogonal orientations. d , Close-up view of the S309 epitope showing the contacts formed with the core fucose (labeled with a star) and the core N-acetyl-glucosamine of the oligosaccharide at position N343. e , Close-up view of the S309 epitope showing the 20-residue long CDRH3 siting atop the S B helix comprising residues 337-344. The oligosaccharide at position N343 is omitted for clarity. In panels ( c-d ), selected residues involved in interactions between S309 and SARS-CoV-2 S are shown. F , Molecular surface representation of the SARS-CoV-2 S trimer showing the S309 footprint colored by residue conservation on one protomer among SARS-CoV-2 and SARS-CoV S glycoproteins. The other two protomers are colored pink and gold.

    Techniques Used: Labeling

    4) Product Images from "CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation"

    Article Title: CoVaccine HT™ adjuvant potentiates robust immune responses to recombinant SARS-CoV-2 Spike S1 immunisation

    Journal: bioRxiv

    doi: 10.1101/2020.07.24.220715

    Detection of IFN-γ secreting cells from mice immunised with SARS-CoV-2 vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/mL or 0.5 μg/mL per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/106 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. *** p ≤ 0.001, **** p ≤ 0.0001.
    Figure Legend Snippet: Detection of IFN-γ secreting cells from mice immunised with SARS-CoV-2 vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunised with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunisations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 hours with PepTivator® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/mL or 0.5 μg/mL per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/106 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. *** p ≤ 0.001, **** p ≤ 0.0001.

    Techniques Used: Mouse Assay, Incubation, Activity Assay

    Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. C Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in panels (B) and (C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads).
    Figure Legend Snippet: Immunogenicity and specificity to SARS-CoV-2 S1 immunisation. A Timeline schematic of BALB/c immunisations and bleeds with a table detailing the study design. B Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. C Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in panels (B) and (C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads).

    Techniques Used: Fluorescence, Binding Assay, Magnetic Beads, Negative Control

    5) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    6) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    7) Product Images from "Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation"

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    Figure Legend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL
    Figure Legend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Techniques Used: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    8) Product Images from "CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1 Immunization"

    Article Title: CoVaccine HT™ Adjuvant Potentiates Robust Immune Responses to Recombinant SARS-CoV-2 Spike S1 Immunization

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.599587

    Immunogenicity and specificity to SARS-CoV-2 S1 immunization. (A) Timeline schematic of BALB/c immunizations and bleeds with a table detailing the study design. (B) Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. (C) Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in (B, C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads). Statistics by standard two-way ANOVA. ****p-value
    Figure Legend Snippet: Immunogenicity and specificity to SARS-CoV-2 S1 immunization. (A) Timeline schematic of BALB/c immunizations and bleeds with a table detailing the study design. (B) Median fluorescence intensity (MFI) of serum antibodies from each group binding to custom magnetic beads coupled with Spike S1 proteins from either SARS-CoV-2 (SARS-2), SARS-CoV (SARS), or MERS-CoV (MERS) on day 14 and 35. (C) Antibody reactivity to SARS-2, SARS, and MERS antigens throughout the study. Graphs in (B, C) are on a logarithmic scale representing geometric mean MFI responses with 95% confidence interval (CI). The dashed lines represent assay cut-off values determined by the mean plus three standard deviations of the negative control (BSA coupled beads). Statistics by standard two-way ANOVA. ****p-value

    Techniques Used: Fluorescence, Binding Assay, Magnetic Beads, Negative Control

    Detection of IFN-γ secreting cells from mice immunized with SARS-CoV-2. vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunized with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunizations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 h with PepTivator ® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/ml or 0.5 μg/ml per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/10 6 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. Significance of differences between groups was determined by one-way ANOVA followed by a “Tukey’s multiple comparison” ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: Detection of IFN-γ secreting cells from mice immunized with SARS-CoV-2. vaccines. The splenocytes were obtained from mice (2 to 3 per group) immunized with SARS-CoV-2 S1 protein, adjuvanted with CoVaccine HT™ or Alum, or S1 protein alone on day 28 (one-week after booster immunizations). Pooled splenocytes obtained from two naïve mice were used as controls. The cells were incubated for 40 h with PepTivator ® SARS-CoV-2 Prot_S1 peptide pools at 0.2 μg/ml or 0.5 μg/ml per peptide or medium. IFN-γ secreting cells were enumerated by FluoroSpot as detailed in the methods section. The results are expressed as the number of spot forming cells (SFC)/10 6 splenocytes after subtraction of the number of spots formed by cells in medium only wells to correct for background activity. Significance of differences between groups was determined by one-way ANOVA followed by a “Tukey’s multiple comparison” ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Mouse Assay, Incubation, Activity Assay

    Related Articles

    other:

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: As shown in (A), these four antibodies have very different affinities towards the S1 protein of SARS-CoV-2.

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: The first antibody, CR3022, is a therapeutic human IgG originally developed against the S1 protein of SARS-CoV.

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: They were also believed to have cross-reactivities with the S1 protein of SARS-CoV-2.

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: The remaining three antibodies, D001, D003, and D006, are humanized chimeric IgGs (the precursors of D001 and D003 were originally raised in mouse and D006 was originally raised in rabbit) that were developed against the S1 protein of SARS-CoV.

    Concentration Assay:

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: Same as in the calibration antibody experiments, we performed a side-by-side study with the recombinant viral antigens from SARS-CoV-2 and SARS-CoV. .. To mimic actual clinical setting, we used 10 times diluted human serum as the solvent of the viral antigen, as we do not expect to see a high concentration of viral S1 protein in serum (or saliva). .. The entire dynamic range of the S1 detection assay is presented in (B).

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    Sino Biological s2 subunit
    RXXR motifs located at the border between S1 and S2 are required for efficient processing of the Middle East respiratory syndrome coronavirus spike protein (MERS-S). A , The domain organization of the MERS-S protein is schematically depicted. The MERS-S sequence at the border between the S1 and S2 subunits is shown. RXXR motifs, which constitute potential cleavage sites, are highlighted, and the predicted start of the <t>S2</t> subunit is underlined. The mutations introduced into the potential cleavage sites in MERS-S are shown. B , 293T cells were transfected with expression plasmids coding for MERS-S wild type and the indicated MERS-S mutants equipped with a C-terminal V5 tag. Transfection of empty plasmid (pcDNA) served as negative control. Expression of S proteins in cell lysates was determined by Western blot, using a V5 tag–specific monoclonal antibody. Expression of β-actin in cell lysates was assessed as a loading control. The results shown are representative for at least 3 independent experiments. Abbreviations: CT, cytoplasmic tail; PCM, potential cleavage site mutant; RBD, receptor binding domain; SP, signal peptide; TM, transmembrane domain.
    S2 Subunit, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological s1 protein
    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the <t>S1</t> protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.
    S1 Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological spike protein of sars cov 2
    Glycolysis and pentose phosphate pathway as host target for antiviral therapy against <t>SARS-CoV-2</t> (A) Heatmap of changes in protein abundance of components of glycolysis and pentose phosphate pathway in SARS-CoV-2 infected Caco-2 cells at 24h post infection. A Z score transformation was performed such that red and blue represent high and low protein abundance respectively. The plot was performed using the heatmaps2 function of the gplots package of the R suite. (B) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT. Caco-2 cells were pre-treated with different concentration of BOT for 24h. The cells were then infected with two different SARS-CoV-2 strains at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Representative images of three independent experiments are shown. (C) Dose-response curves of viral inhibition and cell viability in BOT treated cells. Percentage of viral inhibition was evaluated by spike protein staining and cell viability was measured by MTT assay. The IC50 and CC50 values were determined using the curve regression function of GraphPad Prism 8. Both plots represent mean+SD of three independent experiments performed with three technical replicates. (D) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. The number of SARS-CoV-2/FFM7 RNA was determined by qRT-PCR of RdRp gene and depicted as RNA copies/ml. The bar plot represents mean+SD of three independent experiments performed with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. ns: not significant; * p≤0.05; ** p≤0.01. (E) Inhibition of viral infection in BOT treated cells in combination with 2DG. Caco-2 cells were pre-treated with different concentration of BOT for 24h. Then the 2DG at concentration 5mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Percentage of viral inhibition was evaluated by spike protein staining. Bar graph depicts mean+SD of three independent experiments with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. *** p≤0.005 (F) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Representative images of three independent experiments are shown. (G) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com.
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    RXXR motifs located at the border between S1 and S2 are required for efficient processing of the Middle East respiratory syndrome coronavirus spike protein (MERS-S). A , The domain organization of the MERS-S protein is schematically depicted. The MERS-S sequence at the border between the S1 and S2 subunits is shown. RXXR motifs, which constitute potential cleavage sites, are highlighted, and the predicted start of the S2 subunit is underlined. The mutations introduced into the potential cleavage sites in MERS-S are shown. B , 293T cells were transfected with expression plasmids coding for MERS-S wild type and the indicated MERS-S mutants equipped with a C-terminal V5 tag. Transfection of empty plasmid (pcDNA) served as negative control. Expression of S proteins in cell lysates was determined by Western blot, using a V5 tag–specific monoclonal antibody. Expression of β-actin in cell lysates was assessed as a loading control. The results shown are representative for at least 3 independent experiments. Abbreviations: CT, cytoplasmic tail; PCM, potential cleavage site mutant; RBD, receptor binding domain; SP, signal peptide; TM, transmembrane domain.

    Journal: The Journal of Infectious Diseases

    Article Title: Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity

    doi: 10.1093/infdis/jiu407

    Figure Lengend Snippet: RXXR motifs located at the border between S1 and S2 are required for efficient processing of the Middle East respiratory syndrome coronavirus spike protein (MERS-S). A , The domain organization of the MERS-S protein is schematically depicted. The MERS-S sequence at the border between the S1 and S2 subunits is shown. RXXR motifs, which constitute potential cleavage sites, are highlighted, and the predicted start of the S2 subunit is underlined. The mutations introduced into the potential cleavage sites in MERS-S are shown. B , 293T cells were transfected with expression plasmids coding for MERS-S wild type and the indicated MERS-S mutants equipped with a C-terminal V5 tag. Transfection of empty plasmid (pcDNA) served as negative control. Expression of S proteins in cell lysates was determined by Western blot, using a V5 tag–specific monoclonal antibody. Expression of β-actin in cell lysates was assessed as a loading control. The results shown are representative for at least 3 independent experiments. Abbreviations: CT, cytoplasmic tail; PCM, potential cleavage site mutant; RBD, receptor binding domain; SP, signal peptide; TM, transmembrane domain.

    Article Snippet: MERS-S expression was detected using a monoclonal antibody directed against the V5 tag (Invitrogen) or a polyclonal antibody directed against the S2 subunit of the MERS-S protein (Sino Biological).

    Techniques: Sequencing, Transfection, Expressing, Plasmid Preparation, Negative Control, Western Blot, Mutagenesis, Binding Assay

    The Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein (MERS-S) is cleaved in transfected and infected cells. 293T cells were transfected with a plasmid encoding the MERS-S protein or with empty plasmid (pcDNA). Vero B4 cells were either infected with MERS-CoV at a multiplicity of infection of 5 or mock infected. Subsequently, the cells were lysed and analyzed by Western blot, using a polyclonal antibody directed against the S2 subunit of MERS-S. A β-actin antibody served as a loading control. Similar results were obtained in 2 separate experiments.

    Journal: The Journal of Infectious Diseases

    Article Title: Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity

    doi: 10.1093/infdis/jiu407

    Figure Lengend Snippet: The Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein (MERS-S) is cleaved in transfected and infected cells. 293T cells were transfected with a plasmid encoding the MERS-S protein or with empty plasmid (pcDNA). Vero B4 cells were either infected with MERS-CoV at a multiplicity of infection of 5 or mock infected. Subsequently, the cells were lysed and analyzed by Western blot, using a polyclonal antibody directed against the S2 subunit of MERS-S. A β-actin antibody served as a loading control. Similar results were obtained in 2 separate experiments.

    Article Snippet: MERS-S expression was detected using a monoclonal antibody directed against the V5 tag (Invitrogen) or a polyclonal antibody directed against the S2 subunit of the MERS-S protein (Sino Biological).

    Techniques: Transfection, Infection, Plasmid Preparation, Western Blot

    Comparison of saliva and serum SARS-CoV-2 antigen-specific IgG responses by days post-symptom onset (DPSO). The trajectories of IgG responses (red solid lines) and confidence intervals (semitransparent background) were estimated using a LOESS curve. Dashed red lines indicate cutoff values for each antigen. Sino Biol., Sino Biological; NAC, Native Antigen Company; N, nucleocapsid protein; ECD, ectodomain; S1/S2, S1 or S2 subunit of the spike protein; RBD, receptor binding domain; (h), produced in human cells; (i), produced in insect cells; MFI, median fluorescence intensity; a.u., arbitrary units.

    Journal: Journal of Clinical Microbiology

    Article Title: COVID-19 Serology at Population Scale: SARS-CoV-2-Specific Antibody Responses in Saliva

    doi: 10.1128/JCM.02204-20

    Figure Lengend Snippet: Comparison of saliva and serum SARS-CoV-2 antigen-specific IgG responses by days post-symptom onset (DPSO). The trajectories of IgG responses (red solid lines) and confidence intervals (semitransparent background) were estimated using a LOESS curve. Dashed red lines indicate cutoff values for each antigen. Sino Biol., Sino Biological; NAC, Native Antigen Company; N, nucleocapsid protein; ECD, ectodomain; S1/S2, S1 or S2 subunit of the spike protein; RBD, receptor binding domain; (h), produced in human cells; (i), produced in insect cells; MFI, median fluorescence intensity; a.u., arbitrary units.

    Article Snippet: These included four SARS-CoV-2 receptor binding domain (RBD) proteins, one ectodomain (ECD) protein containing the S1 and S2 subunits of the spike protein, two S1 subunits, one S2 subunit, and two N proteins (see Table S1 in the supplemental material).

    Techniques: Binding Assay, Produced, Fluorescence

    Assay sensitivity and specificity for each SARS-CoV-2 antigen and antibody isotype using saliva (a) and serum (b). Samples collected from individuals with RT-PCR-confirmed prior SARS-CoV-2 infection are stratified by time since symptom onset. Darker shades of green indicate higher and darker shades of red indicate lower sensitivity and specificity. Sino Biol., Sino Biological; NAC, Native Antigen Company; N, nucleocapsid protein; ECD, ectodomain; S1/S2, S1 or S2 subunit of the spike protein; RBD, receptor binding domain; (h), produced in human cells; (i), produced in insect cells.

    Journal: Journal of Clinical Microbiology

    Article Title: COVID-19 Serology at Population Scale: SARS-CoV-2-Specific Antibody Responses in Saliva

    doi: 10.1128/JCM.02204-20

    Figure Lengend Snippet: Assay sensitivity and specificity for each SARS-CoV-2 antigen and antibody isotype using saliva (a) and serum (b). Samples collected from individuals with RT-PCR-confirmed prior SARS-CoV-2 infection are stratified by time since symptom onset. Darker shades of green indicate higher and darker shades of red indicate lower sensitivity and specificity. Sino Biol., Sino Biological; NAC, Native Antigen Company; N, nucleocapsid protein; ECD, ectodomain; S1/S2, S1 or S2 subunit of the spike protein; RBD, receptor binding domain; (h), produced in human cells; (i), produced in insect cells.

    Article Snippet: These included four SARS-CoV-2 receptor binding domain (RBD) proteins, one ectodomain (ECD) protein containing the S1 and S2 subunits of the spike protein, two S1 subunits, one S2 subunit, and two N proteins (see Table S1 in the supplemental material).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Binding Assay, Produced

    Correlation between matched SARS-CoV-2-specific IgG responses in saliva and serum ( n = 28). The Pearson correlation coefficient is provided for IgG responses to each antigen. Sino Biol., Sino Biological; NAC, Native Antigen Company; N, nucleocapsid protein; ECD, ectodomain; S1/S2, S1 or S2 subunit of the spike protein; RBD, receptor binding domain; (h), produced in human cells; (i), produced in insect cells; MFI, median fluorescence intensity.

    Journal: Journal of Clinical Microbiology

    Article Title: COVID-19 Serology at Population Scale: SARS-CoV-2-Specific Antibody Responses in Saliva

    doi: 10.1128/JCM.02204-20

    Figure Lengend Snippet: Correlation between matched SARS-CoV-2-specific IgG responses in saliva and serum ( n = 28). The Pearson correlation coefficient is provided for IgG responses to each antigen. Sino Biol., Sino Biological; NAC, Native Antigen Company; N, nucleocapsid protein; ECD, ectodomain; S1/S2, S1 or S2 subunit of the spike protein; RBD, receptor binding domain; (h), produced in human cells; (i), produced in insect cells; MFI, median fluorescence intensity.

    Article Snippet: These included four SARS-CoV-2 receptor binding domain (RBD) proteins, one ectodomain (ECD) protein containing the S1 and S2 subunits of the spike protein, two S1 subunits, one S2 subunit, and two N proteins (see Table S1 in the supplemental material).

    Techniques: Binding Assay, Produced, Fluorescence

    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Journal: Biosensors & Bioelectronics

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV-2. (B) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from SARS-CoV (B). The solid lines are the linear fit of the data in the log-log scale. D006 is the only antibody that has a high affinity and high specificity towards SARS-CoV-2 S1. Illustration of the assay mechanism, which uses a single-step ELISA format, is shown in Fig. 1 (A). The sample-to-answer time of this assay is 8 min.

    Article Snippet: The remaining three antibodies, D001, D003, and D006, are humanized chimeric IgGs (the precursors of D001 and D003 were originally raised in mouse and D006 was originally raised in rabbit) that were developed against the S1 protein of SARS-CoV.

    Techniques: Enzyme-linked Immunosorbent Assay

    SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Journal: Biosensors & Bioelectronics

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: SARS-CoV-2 antigen detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 40 min. (B) Entire dynamic ranges of SARS-CoV-2 S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein is 0.004 ng/mL

    Article Snippet: The remaining three antibodies, D001, D003, and D006, are humanized chimeric IgGs (the precursors of D001 and D003 were originally raised in mouse and D006 was originally raised in rabbit) that were developed against the S1 protein of SARS-CoV.

    Techniques: Standard Deviation

    Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Biosensors & Bioelectronics

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: Evaluation of anti-S1 calibration antibodies. (A) Entire dynamic ranges for the detection of the four humanized monoclonal antibodies (against SARS-CoV-2 S1). The concentrations were prepared from 3 times of serial dilution (starting from 4800 ng/mL). The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3 × standard deviation of the background. (B) Comparison of the linear dynamic ranges. (C)–(F) Detection of the calibration antibodies in 50 times diluted serum, against the S1 protein from SARS-CoV-2 (red squares) and SARS-CoV (black circles). The calibration curves are generated with three different monoclonal humanized antibodies (CR3022 in (C), D001 in (D), D003 in (E), and D006 in (D)). The solid lines are the linear fit for the data in the log-log scale. Error bars are generated from duplicate measurements. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: The remaining three antibodies, D001, D003, and D006, are humanized chimeric IgGs (the precursors of D001 and D003 were originally raised in mouse and D006 was originally raised in rabbit) that were developed against the S1 protein of SARS-CoV.

    Techniques: Serial Dilution, Standard Deviation, Generated

    Glycolysis and pentose phosphate pathway as host target for antiviral therapy against SARS-CoV-2 (A) Heatmap of changes in protein abundance of components of glycolysis and pentose phosphate pathway in SARS-CoV-2 infected Caco-2 cells at 24h post infection. A Z score transformation was performed such that red and blue represent high and low protein abundance respectively. The plot was performed using the heatmaps2 function of the gplots package of the R suite. (B) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT. Caco-2 cells were pre-treated with different concentration of BOT for 24h. The cells were then infected with two different SARS-CoV-2 strains at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Representative images of three independent experiments are shown. (C) Dose-response curves of viral inhibition and cell viability in BOT treated cells. Percentage of viral inhibition was evaluated by spike protein staining and cell viability was measured by MTT assay. The IC50 and CC50 values were determined using the curve regression function of GraphPad Prism 8. Both plots represent mean+SD of three independent experiments performed with three technical replicates. (D) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. The number of SARS-CoV-2/FFM7 RNA was determined by qRT-PCR of RdRp gene and depicted as RNA copies/ml. The bar plot represents mean+SD of three independent experiments performed with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. ns: not significant; * p≤0.05; ** p≤0.01. (E) Inhibition of viral infection in BOT treated cells in combination with 2DG. Caco-2 cells were pre-treated with different concentration of BOT for 24h. Then the 2DG at concentration 5mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Percentage of viral inhibition was evaluated by spike protein staining. Bar graph depicts mean+SD of three independent experiments with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. *** p≤0.005 (F) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Representative images of three independent experiments are shown. (G) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com.

    Journal: bioRxiv

    Article Title: Targeting pentose phosphate pathway for SARS-CoV-2 therapy

    doi: 10.1101/2020.08.19.257022

    Figure Lengend Snippet: Glycolysis and pentose phosphate pathway as host target for antiviral therapy against SARS-CoV-2 (A) Heatmap of changes in protein abundance of components of glycolysis and pentose phosphate pathway in SARS-CoV-2 infected Caco-2 cells at 24h post infection. A Z score transformation was performed such that red and blue represent high and low protein abundance respectively. The plot was performed using the heatmaps2 function of the gplots package of the R suite. (B) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM1 and SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT. Caco-2 cells were pre-treated with different concentration of BOT for 24h. The cells were then infected with two different SARS-CoV-2 strains at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Representative images of three independent experiments are shown. (C) Dose-response curves of viral inhibition and cell viability in BOT treated cells. Percentage of viral inhibition was evaluated by spike protein staining and cell viability was measured by MTT assay. The IC50 and CC50 values were determined using the curve regression function of GraphPad Prism 8. Both plots represent mean+SD of three independent experiments performed with three technical replicates. (D) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. The number of SARS-CoV-2/FFM7 RNA was determined by qRT-PCR of RdRp gene and depicted as RNA copies/ml. The bar plot represents mean+SD of three independent experiments performed with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. ns: not significant; * p≤0.05; ** p≤0.01. (E) Inhibition of viral infection in BOT treated cells in combination with 2DG. Caco-2 cells were pre-treated with different concentration of BOT for 24h. Then the 2DG at concentration 5mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. 24h post infection, cells were fixed and stain for spike protein. Percentage of viral inhibition was evaluated by spike protein staining. Bar graph depicts mean+SD of three independent experiments with three technical replicates. Statistical significance was determined with a two sided unpaired t-test. *** p≤0.005 (F) Immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Representative images of three independent experiments are shown. (G) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com.

    Article Snippet: 24h post infection, cells were fixed with acetone:methanol (40:60) solution followed by incubation with a primary monoclonal antibody directed against the spike protein of SARS-CoV-2 (1:1500, Sinobiological).

    Techniques: Infection, Transformation Assay, Immunohistochemistry, Staining, Concentration Assay, Inhibition, MTT Assay, Quantitative RT-PCR