sars cov 2 spike  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD AVI His Recombinant Protein Biotinylated 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 a c terminal polyhistidine tagged AVI tag at the C terminus The purified protein was biotinylated in vitro
    Catalog Number:
    40592-V27B-B
    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:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological sars cov 2 spike
    Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of <t>SARS-CoV-2,</t> and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with a c terminal polyhistidine tagged AVI tag at the C terminus The purified protein was biotinylated in vitro
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    sars cov 2 spike - by Bioz Stars, 2021-04
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    1) Product Images from "A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry"

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21213-4

    Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of SARS-CoV-2, and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR
    Figure Legend Snippet: Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of SARS-CoV-2, and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR

    Techniques Used: Genome Wide, CRISPR, Expressing, Knock-Out, Infection, Sequencing

    Host genes that regulate the surface expression of receptor ACE2 are identified. a The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37 °C for internalization. Viral RNA was extracted for RT-qPCR analysis (two experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). b , c Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody (2 experiments; n = 7 ( b ) or 6 ( c ); one-way ANOVA with Dunnett’s test; mean ± s.d.). d , e Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for western blotting. One representative blot was shown ( d ) and data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments ( e ) (four experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). f , g The impact on viral production in CCDC53 gene-edited Calu-3 cells. The mixed cell population was infected with Sfull ( f ) or Sdel ( g ) to assess the virus yield (two experiments; n = 6; two-way ANOVA with Sidak’s test). * P
    Figure Legend Snippet: Host genes that regulate the surface expression of receptor ACE2 are identified. a The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37 °C for internalization. Viral RNA was extracted for RT-qPCR analysis (two experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). b , c Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody (2 experiments; n = 7 ( b ) or 6 ( c ); one-way ANOVA with Dunnett’s test; mean ± s.d.). d , e Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for western blotting. One representative blot was shown ( d ) and data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments ( e ) (four experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). f , g The impact on viral production in CCDC53 gene-edited Calu-3 cells. The mixed cell population was infected with Sfull ( f ) or Sdel ( g ) to assess the virus yield (two experiments; n = 6; two-way ANOVA with Sidak’s test). * P

    Techniques Used: Expressing, Binding Assay, Incubation, Quantitative RT-PCR, Flow Cytometry, Recombinant, Labeling, Immunoprecipitation, Western Blot, Infection

    Genes identified are required for the endosomal cell entry of SARS-CoV-2, SARS-CoV, and MERS-CoV. a–d The selected genes were verified for the infection by pseudovirus bearing the spike protein of SARS-CoV-2 Sdel strain ( a ), the the glycoprotein of vesicular stomatitis virus (VSV-G) ( b ), the spike protein of SARS-CoV ( c ), or the spike protein of MERS-CoV ( d ) (two experiments; n = 4–11; one-way ANOVA with Dunnett’s test; mean ± s.d.). One representative sgRNA per gene was used in A549-ACE2 cells. e The genes selected were verified for the infection by the SARS-CoV-2 Sfull live virus (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). f Effect of NPC1 inhibitor U18666A on virus infection. Cells were treated with U18666A at the indicated concentrations 2 h prior to or 2 h post infection by Sfull or Sdel live virus. The viral N-positive cells were calculated (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the controls of individual experiments. ** P
    Figure Legend Snippet: Genes identified are required for the endosomal cell entry of SARS-CoV-2, SARS-CoV, and MERS-CoV. a–d The selected genes were verified for the infection by pseudovirus bearing the spike protein of SARS-CoV-2 Sdel strain ( a ), the the glycoprotein of vesicular stomatitis virus (VSV-G) ( b ), the spike protein of SARS-CoV ( c ), or the spike protein of MERS-CoV ( d ) (two experiments; n = 4–11; one-way ANOVA with Dunnett’s test; mean ± s.d.). One representative sgRNA per gene was used in A549-ACE2 cells. e The genes selected were verified for the infection by the SARS-CoV-2 Sfull live virus (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). f Effect of NPC1 inhibitor U18666A on virus infection. Cells were treated with U18666A at the indicated concentrations 2 h prior to or 2 h post infection by Sfull or Sdel live virus. The viral N-positive cells were calculated (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the controls of individual experiments. ** P

    Techniques Used: Infection

    The deletion at the S1/S2 boundary of spike protein propels the virus to enter cells through the endosomal pathway. a Sequence alignment of spike protein encompassing the cleavage site between S1 and S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel strain) deletion were used to compare with that of SARS-CoV. The insertion of multi-basic amino acids in spike protein of SARS-CoV-2 was shown in red. b Comparison of the replication property between Sfull and Sdel strains in different cell lines. The percentage of nucleocapsid (N) protein-positive cells was analyzed by imaging-based analysis following virus infection (two or more experiments; n = 6 except for Calu-3 in which n = 8; one-way ANOVA with Dunnett’s test; mean ± s.d.). c Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data are normalized to the Sfull of individual experiments (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). d Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines (two experiments; n = 4 or 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the untreated group of individual experiments. **** P
    Figure Legend Snippet: The deletion at the S1/S2 boundary of spike protein propels the virus to enter cells through the endosomal pathway. a Sequence alignment of spike protein encompassing the cleavage site between S1 and S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel strain) deletion were used to compare with that of SARS-CoV. The insertion of multi-basic amino acids in spike protein of SARS-CoV-2 was shown in red. b Comparison of the replication property between Sfull and Sdel strains in different cell lines. The percentage of nucleocapsid (N) protein-positive cells was analyzed by imaging-based analysis following virus infection (two or more experiments; n = 6 except for Calu-3 in which n = 8; one-way ANOVA with Dunnett’s test; mean ± s.d.). c Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data are normalized to the Sfull of individual experiments (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). d Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines (two experiments; n = 4 or 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the untreated group of individual experiments. **** P

    Techniques Used: Sequencing, Imaging, Infection, Mutagenesis, Protease Inhibitor

    2) Product Images from "Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants"

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    Journal: bioRxiv

    doi: 10.1101/2021.03.16.435594

    SARS-CoV-2 specific T cell immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD4 + T cells. ( B ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD4 + effector memory T cells (CD44 + CD62L - ) in splenocytes. ( C ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD8 + T. ( D ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD8 + effector memory T cells (CD44 + CD62L - ) in splenocytes. Results were pooled from two independent experiments ( B and D ). Data are presented as the mean ± S.E.M. in C and D, n = 3 or 4 for each group. Each symbol represents an individual mouse.
    Figure Legend Snippet: SARS-CoV-2 specific T cell immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD4 + T cells. ( B ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD4 + effector memory T cells (CD44 + CD62L - ) in splenocytes. ( C ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD8 + T. ( D ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD8 + effector memory T cells (CD44 + CD62L - ) in splenocytes. Results were pooled from two independent experiments ( B and D ). Data are presented as the mean ± S.E.M. in C and D, n = 3 or 4 for each group. Each symbol represents an individual mouse.

    Techniques Used: Mouse Assay, FACS, Staining

    The FACS chromatogram of the competitive inhibition of SARS-CoV-2 pseudovirus infection (harboring EGFP reporter) by the circRNA RBD -translated RBD antigens.
    Figure Legend Snippet: The FACS chromatogram of the competitive inhibition of SARS-CoV-2 pseudovirus infection (harboring EGFP reporter) by the circRNA RBD -translated RBD antigens.

    Techniques Used: FACS, Inhibition, Infection

    The susceptibility of SARS-CoV-2 D614G, B.1.1.7 or B.1.351variants to neutralizing antibodies elicited by the circRNA RBD or circRNA RBD-501Y.V2 vaccines in mice. ( A ) Schematic diagram of circRNA RBD-501Y.V2 circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD-501Y.V2, the RBD antigen harboring the K417N-E484K-N501Y mutations in SARS-CoV-2 501Y.V2 variant. ( B ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data was shown as the mean ± S.E.M. Each symbol represents an individual mouse. ( C ) Sigmodal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD-501Y.V2 (50 μg) immunized mice were collected at 1 week or 2 weeks post boost. The data were shown as the mean ± S.E.M. ( D ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD vaccines. The serum samples were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( E ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD-501Y.V2 vaccines. The serum samples were collected at 1 week post boost. The data were shown as the mean ± S.E.M. (n = 5).
    Figure Legend Snippet: The susceptibility of SARS-CoV-2 D614G, B.1.1.7 or B.1.351variants to neutralizing antibodies elicited by the circRNA RBD or circRNA RBD-501Y.V2 vaccines in mice. ( A ) Schematic diagram of circRNA RBD-501Y.V2 circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD-501Y.V2, the RBD antigen harboring the K417N-E484K-N501Y mutations in SARS-CoV-2 501Y.V2 variant. ( B ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data was shown as the mean ± S.E.M. Each symbol represents an individual mouse. ( C ) Sigmodal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD-501Y.V2 (50 μg) immunized mice were collected at 1 week or 2 weeks post boost. The data were shown as the mean ± S.E.M. ( D ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD vaccines. The serum samples were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( E ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD-501Y.V2 vaccines. The serum samples were collected at 1 week post boost. The data were shown as the mean ± S.E.M. (n = 5).

    Techniques Used: Mouse Assay, Sequencing, Variant Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Neutralization

    Expression of SARS-CoV-2 neutralizing nanobodies or hACE2 decoys via circRNA platform. ( A ) Schematic diagram of circRNA nAB or circRNA hACE2 decoys circularization by the Group I ribozyme autocatalysis. ( B ) Lentivirial-based pseudovirus neutralization assay with the supernatant from cells transfected with circRNA encoding neutralizing nanobodies nAB1, nAB1-Tri, nAB2, nAB2-Tri, nAB3 and nAB3-Tri or ACE2 decoys. The luciferase value was normalized to the circRNA EGFP control. The data was shown as the mean ± S.E.M. (n = 2).
    Figure Legend Snippet: Expression of SARS-CoV-2 neutralizing nanobodies or hACE2 decoys via circRNA platform. ( A ) Schematic diagram of circRNA nAB or circRNA hACE2 decoys circularization by the Group I ribozyme autocatalysis. ( B ) Lentivirial-based pseudovirus neutralization assay with the supernatant from cells transfected with circRNA encoding neutralizing nanobodies nAB1, nAB1-Tri, nAB2, nAB2-Tri, nAB3 and nAB3-Tri or ACE2 decoys. The luciferase value was normalized to the circRNA EGFP control. The data was shown as the mean ± S.E.M. (n = 2).

    Techniques Used: Expressing, Neutralization, Transfection, Luciferase

    Humoral immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) Schematic representation of LNP-circRNA complex. ( B ) Representative of concentration-size graph of LNP-circRNA RBD measured by dynamic light scattering method. ( C ) Schematic diagram of the LNP-circRNA RBD vaccination process in BALB/c mice and serum collection schedule for specific antibodies analysis. ( D ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data were shown as the mean ± S.E.M. (n = 4 or 5). ( E ) Sigmoidal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 2 weeks post the second dose. The data was shown as the mean ± S.E.M. (n = 4). ( F ) Sigmoldal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( G ) The NT50 was calculated using lentivirus-based SARS-CoV-2 pseudovirus. The data was shown as the mean ± S.E.M. (n = 5).
    Figure Legend Snippet: Humoral immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) Schematic representation of LNP-circRNA complex. ( B ) Representative of concentration-size graph of LNP-circRNA RBD measured by dynamic light scattering method. ( C ) Schematic diagram of the LNP-circRNA RBD vaccination process in BALB/c mice and serum collection schedule for specific antibodies analysis. ( D ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data were shown as the mean ± S.E.M. (n = 4 or 5). ( E ) Sigmoidal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 2 weeks post the second dose. The data was shown as the mean ± S.E.M. (n = 4). ( F ) Sigmoldal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( G ) The NT50 was calculated using lentivirus-based SARS-CoV-2 pseudovirus. The data was shown as the mean ± S.E.M. (n = 5).

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

    Flow panel and gating strategy to quantify SARS-CoV-2-RBD-specific T cells in mice. The plots showed the gating strategy of single and viable T cells from spleens. CD4 + or CD8 + T cells were further analyzed with the expression of CD44 and CD62L.
    Figure Legend Snippet: Flow panel and gating strategy to quantify SARS-CoV-2-RBD-specific T cells in mice. The plots showed the gating strategy of single and viable T cells from spleens. CD4 + or CD8 + T cells were further analyzed with the expression of CD44 and CD62L.

    Techniques Used: Mouse Assay, Expressing

    Expression of trimeric SARS-CoV-2 RBD antigens with circular RNAs in vitro . ( A ) Schematic diagram of circRNA RBD circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD, the receptor binding domain of SARS-CoV-2 Spike protein. The arrows indicate the the design of primers for PCR analysis. ( B ) The agarose gel electrophoresis result of the PCR products of linear RNA RBD and circRNA RBD . ( C ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD . The circRNA EGFP and linear RNA RBD were set as controls. ( D ) The quantitative ELISA assay to measure the concentration of RBD antigens in the supernatant. The data in ( B ) was shown as the mean ± S.E.M. (n = 3). ( E ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of mouse NIH3T3 cells transfected with circRNA RBD . The circRNA EGFP was set as controls. ( F ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD for different shelf time (3, 7 or 14 days) at room temperature (∼25°C). ( G ) Quantification of the competitive inhibition of SARS-CoV-2 pseudovirus infection (EGFP) by the circRNA RBD -translated RBD antigens. The circRNA EGFP and linear RNA RBD were set as controls. The data in ( E ) was shown as the mean ± S.E.M. (n = 2).
    Figure Legend Snippet: Expression of trimeric SARS-CoV-2 RBD antigens with circular RNAs in vitro . ( A ) Schematic diagram of circRNA RBD circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD, the receptor binding domain of SARS-CoV-2 Spike protein. The arrows indicate the the design of primers for PCR analysis. ( B ) The agarose gel electrophoresis result of the PCR products of linear RNA RBD and circRNA RBD . ( C ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD . The circRNA EGFP and linear RNA RBD were set as controls. ( D ) The quantitative ELISA assay to measure the concentration of RBD antigens in the supernatant. The data in ( B ) was shown as the mean ± S.E.M. (n = 3). ( E ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of mouse NIH3T3 cells transfected with circRNA RBD . The circRNA EGFP was set as controls. ( F ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD for different shelf time (3, 7 or 14 days) at room temperature (∼25°C). ( G ) Quantification of the competitive inhibition of SARS-CoV-2 pseudovirus infection (EGFP) by the circRNA RBD -translated RBD antigens. The circRNA EGFP and linear RNA RBD were set as controls. The data in ( E ) was shown as the mean ± S.E.M. (n = 2).

    Techniques Used: Expressing, In Vitro, Sequencing, Binding Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition, Infection

    Identification of IL-4 producing CD4 + T cells in mice immunized with SARS-CoV-2 circRNA RBD vaccines. Splenocytes were stimulated with SARS-CoV-2-RBD peptides for 7 hr in the presence of BFA and Monensin. PMA and Ionomycin stimulation were applied as a positive control. Cells were gated on single and viable CD4 + T cells. The plots are representative for two independent experiments with same results.
    Figure Legend Snippet: Identification of IL-4 producing CD4 + T cells in mice immunized with SARS-CoV-2 circRNA RBD vaccines. Splenocytes were stimulated with SARS-CoV-2-RBD peptides for 7 hr in the presence of BFA and Monensin. PMA and Ionomycin stimulation were applied as a positive control. Cells were gated on single and viable CD4 + T cells. The plots are representative for two independent experiments with same results.

    Techniques Used: Mouse Assay, Positive Control

    3) Product Images from "CD47 as a potential biomarker for the early diagnosis of severe COVID-19"

    Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

    Journal: bioRxiv

    doi: 10.1101/2021.03.01.433404

    Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.
    Figure Legend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

    Techniques Used: Derivative Assay, Infection

    SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from [ Bojkova et al., 2020 ]. P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from [ Blanco-Melo et al., 2020 ]). P-values were determined by two-sided Student’s t-test.
    Figure Legend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from [ Bojkova et al., 2020 ]. P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from [ Blanco-Melo et al., 2020 ]). P-values were determined by two-sided Student’s t-test.

    Techniques Used: Infection, Derivative Assay, Polymerase Chain Reaction

    4) Product Images from "Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19"

    Article Title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

    Journal: bioRxiv

    doi: 10.1101/2021.03.19.436183

    Neutralizing antibody titers and RBD binding antibodies of convalescent plasma and hCoV-2IG against various SARS-CoV-2 strains. (A) SARS-CoV-2 neutralizing antibody titers in CP, 2019-IVIG and hCoV-2IG preparations as determined by pseudovirus neutralization assay in 293-ACE2-TMPRSS2 cells with SARS-CoV-2 WA-1 strain, CA variant (B.1.429), UK variant (B.1.1.7), JP variant (P.1) or SA variant (B.1.351). PsVNA50 (50% neutralization titer) and PsVNA80 (80% neutralization titer) titers for control pre-pandemic 2019-IVIG (n=16), convalescent plasma (n =9) and hCoV-2IG (n = 6) were calculated with GraphPad prism version 8. Data show mean values + SEM for PsVNA50 and PsVNA80 titers for each of the 3 antibody groups against the SARS-CoV-2 WA-1, CA, UK, JP and SA variants. (B) End-point virus neutralization titers for six hCoV-2IG lots using wild type authentic SARS-CoV-2 WA-1, UK and SA virus strains in a classical BSL3 neutralization assay based on a plaque assay was performed as described in Materials and Methods. (C) Pearson two-tailed correlations are reported for the calculation of correlation of PRNT50 titers against wild-type SARS-CoV-2 strains (WA-1, UK or SA) and PsVNA50 titers against corresponding pseudovirions expressing either WA-1, UK or SA spike in pseudovirion neutralization assays for the six hCOV-2IG lots. (D) Antibody concentration (in mg/mL) required for each of the six hCoV-2IG batches to achieve 50% neutralization of SARS-CoV-2 WA-1, CA, UK, JP or SA variants in PsVNA. (E-F) Fold-decrease in PsVNA50 neutralization titers against emerging variant strain CA (B.1.429), UK (B.1.1.7), JP (P.1) and SA (B.1.351) for six hCoV-2IG lots (E) and nine CP lots (F) in comparison with SARS-CoV-2 WA-1 strain. The numbers above the group shows the mean fold-change for each variant. (G-H) Total antibody binding (Max RU) of 1mg/mL for the six batches of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6) to purified WA-1 RBD (RBD-wt) and RBD mutants: RBD-K417N, RBD-N501Y and RBD-E484K by SPR (G). The numbers above the group show the mean antibody binding for each RBD. (H) Fold-decrease in antibody binding to mutants RBD-K417N, RBD-N501Y and RBD-E484K of hCoV-2IG in comparison with RBD-wt from WA-1 strain calculated from the data in Panel G. The numbers above the group shows the mean fold-change for each mutant RBD. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was
    Figure Legend Snippet: Neutralizing antibody titers and RBD binding antibodies of convalescent plasma and hCoV-2IG against various SARS-CoV-2 strains. (A) SARS-CoV-2 neutralizing antibody titers in CP, 2019-IVIG and hCoV-2IG preparations as determined by pseudovirus neutralization assay in 293-ACE2-TMPRSS2 cells with SARS-CoV-2 WA-1 strain, CA variant (B.1.429), UK variant (B.1.1.7), JP variant (P.1) or SA variant (B.1.351). PsVNA50 (50% neutralization titer) and PsVNA80 (80% neutralization titer) titers for control pre-pandemic 2019-IVIG (n=16), convalescent plasma (n =9) and hCoV-2IG (n = 6) were calculated with GraphPad prism version 8. Data show mean values + SEM for PsVNA50 and PsVNA80 titers for each of the 3 antibody groups against the SARS-CoV-2 WA-1, CA, UK, JP and SA variants. (B) End-point virus neutralization titers for six hCoV-2IG lots using wild type authentic SARS-CoV-2 WA-1, UK and SA virus strains in a classical BSL3 neutralization assay based on a plaque assay was performed as described in Materials and Methods. (C) Pearson two-tailed correlations are reported for the calculation of correlation of PRNT50 titers against wild-type SARS-CoV-2 strains (WA-1, UK or SA) and PsVNA50 titers against corresponding pseudovirions expressing either WA-1, UK or SA spike in pseudovirion neutralization assays for the six hCOV-2IG lots. (D) Antibody concentration (in mg/mL) required for each of the six hCoV-2IG batches to achieve 50% neutralization of SARS-CoV-2 WA-1, CA, UK, JP or SA variants in PsVNA. (E-F) Fold-decrease in PsVNA50 neutralization titers against emerging variant strain CA (B.1.429), UK (B.1.1.7), JP (P.1) and SA (B.1.351) for six hCoV-2IG lots (E) and nine CP lots (F) in comparison with SARS-CoV-2 WA-1 strain. The numbers above the group shows the mean fold-change for each variant. (G-H) Total antibody binding (Max RU) of 1mg/mL for the six batches of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6) to purified WA-1 RBD (RBD-wt) and RBD mutants: RBD-K417N, RBD-N501Y and RBD-E484K by SPR (G). The numbers above the group show the mean antibody binding for each RBD. (H) Fold-decrease in antibody binding to mutants RBD-K417N, RBD-N501Y and RBD-E484K of hCoV-2IG in comparison with RBD-wt from WA-1 strain calculated from the data in Panel G. The numbers above the group shows the mean fold-change for each mutant RBD. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Techniques Used: Binding Assay, Neutralization, Variant Assay, Plaque Assay, Two Tailed Test, Expressing, Concentration Assay, Purification, SPR Assay, Mutagenesis

    SARS-CoV-2 spike antibody epitope repertoires recognized by hCoV-2IG. SARS-CoV-2 spike GFPDL analyses of IgG antibodies in six batches of hCoV-2IG. (A) Number of IgG bound phage clones selected using SARS-CoV-2 spike GFPDL on six lots of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6). (B) Epitope repertoires of IgG antibody in hCoV-2IG batches and their alignment to the spike protein of SARS-CoV-2. Graphical distribution of representative clones with a frequency of > 2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the alignment of peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value is shown enclosed in a black box beneath the alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and a similar number of phage clones and epitope repertoire was observed in both phage display analysis. (C) SPR binding of hCOV-2IG (n=6; in red), control pre-pandemic 2019-IVIG (n=16; in black) and convalescent plasma (n=9; in blue) with SARS-CoV-2 spike antigenic site peptides identified using GFPDL analysis in Fig. 1B . The amino acid designation is based on the SARS-CoV-2 spike protein sequence ( Fig. S1 ). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution of CP, and 1mg/mL of 2019-IVIG or hCoV-2IG. The numbers above the peptides show the mean value for each respective group antibody binding to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue). All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was
    Figure Legend Snippet: SARS-CoV-2 spike antibody epitope repertoires recognized by hCoV-2IG. SARS-CoV-2 spike GFPDL analyses of IgG antibodies in six batches of hCoV-2IG. (A) Number of IgG bound phage clones selected using SARS-CoV-2 spike GFPDL on six lots of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6). (B) Epitope repertoires of IgG antibody in hCoV-2IG batches and their alignment to the spike protein of SARS-CoV-2. Graphical distribution of representative clones with a frequency of > 2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the alignment of peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value is shown enclosed in a black box beneath the alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and a similar number of phage clones and epitope repertoire was observed in both phage display analysis. (C) SPR binding of hCOV-2IG (n=6; in red), control pre-pandemic 2019-IVIG (n=16; in black) and convalescent plasma (n=9; in blue) with SARS-CoV-2 spike antigenic site peptides identified using GFPDL analysis in Fig. 1B . The amino acid designation is based on the SARS-CoV-2 spike protein sequence ( Fig. S1 ). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution of CP, and 1mg/mL of 2019-IVIG or hCoV-2IG. The numbers above the peptides show the mean value for each respective group antibody binding to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue). All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Techniques Used: Clone Assay, Selection, Sequencing, Isolation, SPR Assay, Binding Assay

    Multiple sequence alignment of Spike protein of SARS-CoV-2 variants. Multiple sequence alignment of various SARS-CoV-2 variants namely WA-1 strain (QII87782.1), CA variant (B.1.429, EPI_ISL_648527), UK variant (B.1.1.7, QQQ47833.1), JP variant (P.1, QRX39425.1), and SA variant (B.1.351, EPI_ISL_678597) was performed using MAFFT version 7 alignment tool ( https://mafft.cbrc.jp/alignment/software/ ). Mutations in any or all of the variants are indicated with a red outline around each of them. Various domains of the spike protein are also indicated namely S1, S2, RBD and FP domains.
    Figure Legend Snippet: Multiple sequence alignment of Spike protein of SARS-CoV-2 variants. Multiple sequence alignment of various SARS-CoV-2 variants namely WA-1 strain (QII87782.1), CA variant (B.1.429, EPI_ISL_648527), UK variant (B.1.1.7, QQQ47833.1), JP variant (P.1, QRX39425.1), and SA variant (B.1.351, EPI_ISL_678597) was performed using MAFFT version 7 alignment tool ( https://mafft.cbrc.jp/alignment/software/ ). Mutations in any or all of the variants are indicated with a red outline around each of them. Various domains of the spike protein are also indicated namely S1, S2, RBD and FP domains.

    Techniques Used: Sequencing, Variant Assay, Software

    SARS-CoV-2 epitope profile of six hCoV-2IG batches. Heat map of immunodominant sites (≥3 clonal frequency in at least one hCoV-2IG lot) on the SARS-CoV-2 spike recognized by IgG antibodies in six hCoV-2IG lots identified using GFPDL analyses. The immunodominant sites on the left indicate amino acid residue of the antigenic sites in the spike protein. Color scale on the right represents range of percentage of clonal occurrences (frequency) of each site. Heat map was generated using R package.
    Figure Legend Snippet: SARS-CoV-2 epitope profile of six hCoV-2IG batches. Heat map of immunodominant sites (≥3 clonal frequency in at least one hCoV-2IG lot) on the SARS-CoV-2 spike recognized by IgG antibodies in six hCoV-2IG lots identified using GFPDL analyses. The immunodominant sites on the left indicate amino acid residue of the antigenic sites in the spike protein. Color scale on the right represents range of percentage of clonal occurrences (frequency) of each site. Heat map was generated using R package.

    Techniques Used: Generated

    5) Product Images from "Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity"

    Article Title: Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity

    Journal: Nature Communications

    doi: 10.1038/s41467-021-20973-3

    Combination of 2 spike protein variants and isotype profiling by multiplex assay increases accuracy to identify SARS-CoV-2 antibody-positive individuals. a , b Scatterplot detailing MultiCoV-Ab cut-offs. Signal to cut-off (S/CO) values are displayed for Spike Trimer against RBD on a logarithmic scale. For IgG ( a ), cut-offs are visualized by straight lines and SARS-CoV-2-infected and uninfected samples are separated by color (black circles – SARS-CoV-2-uninfected; red circles – SARS-CoV-2-infected). For IgA ( b ) cut-offs are visualized as dashed lines and S/CO of 2 used for the combined cut-off is shown as straight lines. SARS-CoV-2-infected samples are split into IgG-positives and -negatives by color as indicated in the plot. c , d Scatterplots display IgG response to additional SARS-CoV-2 antigens contained in the MultiCoV-Ab panel: MFI for spike subdomains S1 vs S2 ( c ) or nucleocapsid antigens N vs N-NTD ( d ) are displayed on a logarithmic scale. SARS-CoV-2-uninfected samples are distinguished from SARS-CoV-2-infected and MultiCoV-Ab classification into positives or negatives as indicated by color. Source data are provided as a Source Data file.
    Figure Legend Snippet: Combination of 2 spike protein variants and isotype profiling by multiplex assay increases accuracy to identify SARS-CoV-2 antibody-positive individuals. a , b Scatterplot detailing MultiCoV-Ab cut-offs. Signal to cut-off (S/CO) values are displayed for Spike Trimer against RBD on a logarithmic scale. For IgG ( a ), cut-offs are visualized by straight lines and SARS-CoV-2-infected and uninfected samples are separated by color (black circles – SARS-CoV-2-uninfected; red circles – SARS-CoV-2-infected). For IgA ( b ) cut-offs are visualized as dashed lines and S/CO of 2 used for the combined cut-off is shown as straight lines. SARS-CoV-2-infected samples are split into IgG-positives and -negatives by color as indicated in the plot. c , d Scatterplots display IgG response to additional SARS-CoV-2 antigens contained in the MultiCoV-Ab panel: MFI for spike subdomains S1 vs S2 ( c ) or nucleocapsid antigens N vs N-NTD ( d ) are displayed on a logarithmic scale. SARS-CoV-2-uninfected samples are distinguished from SARS-CoV-2-infected and MultiCoV-Ab classification into positives or negatives as indicated by color. Source data are provided as a Source Data file.

    Techniques Used: Multiplex Assay, Infection

    Correlation of seasonal hCoV and SARS CoV-2 antibody responses. a Correlation of IgG response for the entire sample set ( n = 1176) is visualized as heatmap based on Spearman’s ρ coefficient; dendrogram on the right side displays antigens after hierarchical clustering was performed. b-c, Immune responses (IgG and IgA) towards hCoV S1 ( b ) and N ( c ) proteins are presented as Box-Whisker plots of sample MFI on a logarithmic scale for SARS-CoV-2-infected (red, n = 310) and uninfected (blue, n = 866) individuals. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. d-e, Relative levels of IgG-specific immune response towards hCoV S1 ( d ) and N ( e ) proteins are presented as Box-Whisker plots/strip chart overlays of log-transformed and per-antigen scaled and centered MFI for the sample subsets of Spike Trimer false positives (blue, n = 17) and combined IgG + IgA false negatives (red, n = 31). Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values, excluding outliers as determined by 1.5 times IQR. Source data are provided as a Source Data file.
    Figure Legend Snippet: Correlation of seasonal hCoV and SARS CoV-2 antibody responses. a Correlation of IgG response for the entire sample set ( n = 1176) is visualized as heatmap based on Spearman’s ρ coefficient; dendrogram on the right side displays antigens after hierarchical clustering was performed. b-c, Immune responses (IgG and IgA) towards hCoV S1 ( b ) and N ( c ) proteins are presented as Box-Whisker plots of sample MFI on a logarithmic scale for SARS-CoV-2-infected (red, n = 310) and uninfected (blue, n = 866) individuals. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. d-e, Relative levels of IgG-specific immune response towards hCoV S1 ( d ) and N ( e ) proteins are presented as Box-Whisker plots/strip chart overlays of log-transformed and per-antigen scaled and centered MFI for the sample subsets of Spike Trimer false positives (blue, n = 17) and combined IgG + IgA false negatives (red, n = 31). Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values, excluding outliers as determined by 1.5 times IQR. Source data are provided as a Source Data file.

    Techniques Used: Whisker Assay, Infection, Transformation Assay, Stripping Membranes

    Multiplex-based seroprofiling allows in-depth characterization of SARS-CoV-2 antibody responses. a Kinetic of SARS-CoV-2 antigen-specific IgA and IgG responses is shown for indicated days after symptom onset for six SARS-CoV-2-specific antigens for five different patients. Patients are indicated by color. b , c Samples of SARS-CoV-2-infected individuals were analyzed to identify antigen- and isotype-specific antibody responses based on hospitalization indicating disease severity ( b ) or age ( c ). Data is presented as Box-Whisker plots of sample MFI on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. p -value (Mann–Whitney U test, two-sided) is displayed at the top of the boxes, indicating differences between signal distribution for respective groups. Cut-off values for MultiCoV-Ab classification are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). Source data are provided as a Source Data file.
    Figure Legend Snippet: Multiplex-based seroprofiling allows in-depth characterization of SARS-CoV-2 antibody responses. a Kinetic of SARS-CoV-2 antigen-specific IgA and IgG responses is shown for indicated days after symptom onset for six SARS-CoV-2-specific antigens for five different patients. Patients are indicated by color. b , c Samples of SARS-CoV-2-infected individuals were analyzed to identify antigen- and isotype-specific antibody responses based on hospitalization indicating disease severity ( b ) or age ( c ). Data is presented as Box-Whisker plots of sample MFI on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. p -value (Mann–Whitney U test, two-sided) is displayed at the top of the boxes, indicating differences between signal distribution for respective groups. Cut-off values for MultiCoV-Ab classification are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). Source data are provided as a Source Data file.

    Techniques Used: Multiplex Assay, Infection, Whisker Assay, Transformation Assay, MANN-WHITNEY

    MultiCoV-Ab, a sensitive and specific tool to monitor SARS-CoV-2 antibody responses. a Control sera (blue, n = 72) and sera from individuals with PCR-confirmed SARS-CoV-2 infection (red, n = 205) were screened in a multiplex bead-based assay using Luminex technology (MultiCoV-Ab) to quantify IgG or IgA responses to various antigens. Reactivity towards trimeric SARS-CoV-2 spike protein (Spike Trimer) or SARS-CoV-2 receptor binding domain of spike (RBD) was found to be the best predictor of SARS-CoV-2 infection. Data are presented as Box-Whisker plots of a sample’s median fluorescence intensity (MFI) on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. Cut-off values for classification for single antigens are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). b Sample set from a , was used to compare assay performance of the MultiCoV-Ab using Spike Trimer and RBD antigens with commercially available single analyte SARS-CoV-2 IVD assays which detect total Ig (Elecsys Anti-SARS-CoV-2 (Roche); ADVIA Centaur SARS-CoV-2 Total (COV2T) (Siemens Healthineers)) or IgG (Anti-SARS-CoV-2-ELISA - IgG (Euroimmun)) or IgA (Anti-SARS-CoV-2-ELISA - IgA (Euroimmun)). SARS-CoV-2 infection status of samples based on PCR diagnostic is indicated as SARS-CoV-2 positive or negative. Antibody test results were classified as negative (blue), positive (red), or borderline (gray) as per the manufacturer’s definition. Only samples with divergent antibody test results are shown. c Performance and specifications as stated in the manufacturer’s IVD assay manual. For the manufacturer sensitivity specification, information for samples > 14 days post-infection are presented. Respective sensitivity and specificity values calculated in this study are given with 95% Clopper-Pearson confidence intervals 52 . Positive and negative predictive values (PPV/NPV) were calculated based on a seropositivity of 3%. Source data are provided as a Source Data file.
    Figure Legend Snippet: MultiCoV-Ab, a sensitive and specific tool to monitor SARS-CoV-2 antibody responses. a Control sera (blue, n = 72) and sera from individuals with PCR-confirmed SARS-CoV-2 infection (red, n = 205) were screened in a multiplex bead-based assay using Luminex technology (MultiCoV-Ab) to quantify IgG or IgA responses to various antigens. Reactivity towards trimeric SARS-CoV-2 spike protein (Spike Trimer) or SARS-CoV-2 receptor binding domain of spike (RBD) was found to be the best predictor of SARS-CoV-2 infection. Data are presented as Box-Whisker plots of a sample’s median fluorescence intensity (MFI) on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. Cut-off values for classification for single antigens are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). b Sample set from a , was used to compare assay performance of the MultiCoV-Ab using Spike Trimer and RBD antigens with commercially available single analyte SARS-CoV-2 IVD assays which detect total Ig (Elecsys Anti-SARS-CoV-2 (Roche); ADVIA Centaur SARS-CoV-2 Total (COV2T) (Siemens Healthineers)) or IgG (Anti-SARS-CoV-2-ELISA - IgG (Euroimmun)) or IgA (Anti-SARS-CoV-2-ELISA - IgA (Euroimmun)). SARS-CoV-2 infection status of samples based on PCR diagnostic is indicated as SARS-CoV-2 positive or negative. Antibody test results were classified as negative (blue), positive (red), or borderline (gray) as per the manufacturer’s definition. Only samples with divergent antibody test results are shown. c Performance and specifications as stated in the manufacturer’s IVD assay manual. For the manufacturer sensitivity specification, information for samples > 14 days post-infection are presented. Respective sensitivity and specificity values calculated in this study are given with 95% Clopper-Pearson confidence intervals 52 . Positive and negative predictive values (PPV/NPV) were calculated based on a seropositivity of 3%. Source data are provided as a Source Data file.

    Techniques Used: Polymerase Chain Reaction, Infection, Multiplex Assay, Bead-based Assay, Luminex, Binding Assay, Whisker Assay, Fluorescence, Transformation Assay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

    Analysis of seasonal hCoV high and low responders. a From the entire study population, groups of α- or β-hCoV high and low responders were built as indicated. High responder were defined as samples with above average MFI values for S1 and N-specific IgGs of the respective hCoV clade. Low responders were defined with below MFI values, correspondingly. Responder groups (i) α-hCoV ↑, red, n = 233, (ii) β-hCoV ↑, green, n = 254, (iii) α-hCoV ↓, blue, n = 172 (iv) β-hCoV ↓, purple, n = 210 are shown as Box-Whisker plots of log-transformed and per-antigen scaled and centered MFI values across hCoV N and S1 antigens. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR are depicted as circles. b The over- or under-representation of SARS-CoV-2 responders (SARS-CoV-2 + , n = 279, as determined by positive MultiCoV-Ab classification) within the four sample groups is visualized in Venn diagrams, stochastic significance was calculated using Fisher’s exact test (two-sided). Source data are provided as a Source Data file.
    Figure Legend Snippet: Analysis of seasonal hCoV high and low responders. a From the entire study population, groups of α- or β-hCoV high and low responders were built as indicated. High responder were defined as samples with above average MFI values for S1 and N-specific IgGs of the respective hCoV clade. Low responders were defined with below MFI values, correspondingly. Responder groups (i) α-hCoV ↑, red, n = 233, (ii) β-hCoV ↑, green, n = 254, (iii) α-hCoV ↓, blue, n = 172 (iv) β-hCoV ↓, purple, n = 210 are shown as Box-Whisker plots of log-transformed and per-antigen scaled and centered MFI values across hCoV N and S1 antigens. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR are depicted as circles. b The over- or under-representation of SARS-CoV-2 responders (SARS-CoV-2 + , n = 279, as determined by positive MultiCoV-Ab classification) within the four sample groups is visualized in Venn diagrams, stochastic significance was calculated using Fisher’s exact test (two-sided). Source data are provided as a Source Data file.

    Techniques Used: Whisker Assay, Transformation Assay

    6) Product Images from "Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody"

    Article Title: Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-82833-w

    Binding characterization and neutralization of SARS-CoV-2 by the nanobody W25. ( a) Pulldown of the W25 nanobody. A recombinant Spike RBD domain of the SARS-CoV-2 spike protein or control BSA protein were covalently bound to NHS-sepharose beads. Further, the W25 nanobody was incubated with control and spike RBD beads, washed, and further eluted in LSD lysis buffer (Invitrogen). Original SDS-Page as supplemental Fig. 3 b. ( b) Unfolding profiles of 2 µM SARS-CoV-2 S1, spike RBD in the absence (black) and presence (red) of 2 µM W25, measured with Tycho NT.6. Binding of W25 to spike RBD leads to strong stabilization and shifts the inflection unfolding temperature (T i ) from 52.1 to 66.3 °C. ( c) MST binding curve for the titration of 1 nM fluorescently labeled W25 into a 16-point serial dilution of SARS-CoV-2 S1, Spike RBD (250 nM to 7.6 pM). W25 binds Spike RBD with sub-nanomolar affinity (K d = 295 ± 84 pM). Error bars show the SD calculated from experiments performed in triplicate. ( d) MST competitive curve for 2 nM of fluorescently labeled W25 incubated with 4 nM SARS-CoV-2 RBD, titrated with a 16-point dilution series of hACE2 (1 µM to 30.5 pM). W25 is displaced by hACE2 with nanomolar concentration (EC50 = 33 ± 9 nM). Error bar show the SD calculated from triplicate experiments. ( e) Diagram of W25 and ACE2 competition for RBD of spike of SARS-CoV-2. ( f) Neutralization assay of SARS-CoV-2 life virus D624 variant with nanobody W25, W25 fused to monomeric Fc (W25FcM) and W25 fused to dimeric Fc (W25Fc) and the previously reported nanobodies VHH-72-Fc (monomeric) and VHH-55-Fc (monomeric). The independent experiments were normalized by percentage of neutralization. ( g) Neutralization assay of SARS-CoV-2 life virus D624 under similar condition as in ( g ). ( h) Comparative neutralization values of W25, W25FcM, W25Fc and VHH-72 FcM against SARS-CoV-2 D614 and G614 virus variants. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Binding characterization and neutralization of SARS-CoV-2 by the nanobody W25. ( a) Pulldown of the W25 nanobody. A recombinant Spike RBD domain of the SARS-CoV-2 spike protein or control BSA protein were covalently bound to NHS-sepharose beads. Further, the W25 nanobody was incubated with control and spike RBD beads, washed, and further eluted in LSD lysis buffer (Invitrogen). Original SDS-Page as supplemental Fig. 3 b. ( b) Unfolding profiles of 2 µM SARS-CoV-2 S1, spike RBD in the absence (black) and presence (red) of 2 µM W25, measured with Tycho NT.6. Binding of W25 to spike RBD leads to strong stabilization and shifts the inflection unfolding temperature (T i ) from 52.1 to 66.3 °C. ( c) MST binding curve for the titration of 1 nM fluorescently labeled W25 into a 16-point serial dilution of SARS-CoV-2 S1, Spike RBD (250 nM to 7.6 pM). W25 binds Spike RBD with sub-nanomolar affinity (K d = 295 ± 84 pM). Error bars show the SD calculated from experiments performed in triplicate. ( d) MST competitive curve for 2 nM of fluorescently labeled W25 incubated with 4 nM SARS-CoV-2 RBD, titrated with a 16-point dilution series of hACE2 (1 µM to 30.5 pM). W25 is displaced by hACE2 with nanomolar concentration (EC50 = 33 ± 9 nM). Error bar show the SD calculated from triplicate experiments. ( e) Diagram of W25 and ACE2 competition for RBD of spike of SARS-CoV-2. ( f) Neutralization assay of SARS-CoV-2 life virus D624 variant with nanobody W25, W25 fused to monomeric Fc (W25FcM) and W25 fused to dimeric Fc (W25Fc) and the previously reported nanobodies VHH-72-Fc (monomeric) and VHH-55-Fc (monomeric). The independent experiments were normalized by percentage of neutralization. ( g) Neutralization assay of SARS-CoV-2 life virus D624 under similar condition as in ( g ). ( h) Comparative neutralization values of W25, W25FcM, W25Fc and VHH-72 FcM against SARS-CoV-2 D614 and G614 virus variants. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Binding Assay, Neutralization, Recombinant, Incubation, Lysis, SDS Page, Titration, Labeling, Serial Dilution, Concentration Assay, Variant Assay

    Diagram of W25 neutralization of SARS-CoV-2. Illustration by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Diagram of W25 neutralization of SARS-CoV-2. Illustration by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Neutralization

    Immunization of the spike of SARS-CoV-2 and a simple density gradient method for the selection of nanobodies. (a) SDS-Page to ensure protein integrity of full-length spike of SARS-CoV-2 before immunization. ( b) Adult alpaca immunized with spike . (c) Evaluation of the alpaca´s immune response by dot blot. Image shows the reaction to decreasing amounts of Spike and bovine serum albumin (negative control) using a pre-immunization control, and after one immunization (1 week), or two immunizations (3 weeks) with full-length SARS-CoV-2 spike, using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody. (d) ELISA before and after the second immunization (3 weeks) n = 4 error bars indicate standard deviation statistic t-test, ** P ≤ 0.005. (e) Schematic representation of novel protocol for isolation of nanobodies using density gradient separation. The bacterial display library expressing the nanobodies on the surface of bacteria is briefly incubated with conventional sepharose beads coated with the antigen of interest. Directly after the mixture is deposited on a Ficoll gradient conic tube and centrifuged at 200 g for 1 min, the beads drive through the gradient to the bottom of the tube with the bacteria expressing specific nanobodies, while unbound bacteria remain on the surface of the gradient. The beads are then resuspended, and bacterial clones are isolated for biochemical binding confirmation. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Immunization of the spike of SARS-CoV-2 and a simple density gradient method for the selection of nanobodies. (a) SDS-Page to ensure protein integrity of full-length spike of SARS-CoV-2 before immunization. ( b) Adult alpaca immunized with spike . (c) Evaluation of the alpaca´s immune response by dot blot. Image shows the reaction to decreasing amounts of Spike and bovine serum albumin (negative control) using a pre-immunization control, and after one immunization (1 week), or two immunizations (3 weeks) with full-length SARS-CoV-2 spike, using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody. (d) ELISA before and after the second immunization (3 weeks) n = 4 error bars indicate standard deviation statistic t-test, ** P ≤ 0.005. (e) Schematic representation of novel protocol for isolation of nanobodies using density gradient separation. The bacterial display library expressing the nanobodies on the surface of bacteria is briefly incubated with conventional sepharose beads coated with the antigen of interest. Directly after the mixture is deposited on a Ficoll gradient conic tube and centrifuged at 200 g for 1 min, the beads drive through the gradient to the bottom of the tube with the bacteria expressing specific nanobodies, while unbound bacteria remain on the surface of the gradient. The beads are then resuspended, and bacterial clones are isolated for biochemical binding confirmation. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Selection, SDS Page, Dot Blot, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Isolation, Expressing, Incubation, Clone Assay, Binding Assay

    Dual biochemical and microscopy-based selection of nanobodies. ( a) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25 and W23 as the primary antibody. Mouse anti-Myc (1:3000) followed by anti-mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. Lineal contrast and grey scale were applied to the image, original dot blot scan is shown in the supplemental Fig. 3 a. ( b) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by mouse anti-Myc (1:3000) and anti-mouse-Alexa 647. The image shows two positive clones (W25 and W23), and an example of a negative Nanobody the screening assay was performed once, scale bar indicates 20 µm. ( c) Sequence alignment of aminoacidic sequence of W25 and W23. CDR sequences are marked with a black line. ( d) Purification from periplasm of bacteria, elution fraction of a single liter of bacterial culture n = 5. ( e) Immunodetection as in (b) , using purified protein n = 3, scale bar indicates 20 µm. ( f) ELISA assay of full-length Spike of SARS-CoV-2 using conjugated W25-HRP nanobody n = 3. ( g) ELISA assay of RBD of Spike using W25-HRP conjugate Nanobody n = 3. Statistic t-test, *** P ≤ 0.001; ** P ≤ 0.005; * P ≤ 0.01 to -W25 control. Illustrations (f,g) by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Dual biochemical and microscopy-based selection of nanobodies. ( a) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25 and W23 as the primary antibody. Mouse anti-Myc (1:3000) followed by anti-mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. Lineal contrast and grey scale were applied to the image, original dot blot scan is shown in the supplemental Fig. 3 a. ( b) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by mouse anti-Myc (1:3000) and anti-mouse-Alexa 647. The image shows two positive clones (W25 and W23), and an example of a negative Nanobody the screening assay was performed once, scale bar indicates 20 µm. ( c) Sequence alignment of aminoacidic sequence of W25 and W23. CDR sequences are marked with a black line. ( d) Purification from periplasm of bacteria, elution fraction of a single liter of bacterial culture n = 5. ( e) Immunodetection as in (b) , using purified protein n = 3, scale bar indicates 20 µm. ( f) ELISA assay of full-length Spike of SARS-CoV-2 using conjugated W25-HRP nanobody n = 3. ( g) ELISA assay of RBD of Spike using W25-HRP conjugate Nanobody n = 3. Statistic t-test, *** P ≤ 0.001; ** P ≤ 0.005; * P ≤ 0.01 to -W25 control. Illustrations (f,g) by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Microscopy, Selection, Dot Blot, Immunodetection, Clone Assay, Negative Control, Transfection, Screening Assay, Sequencing, Purification, Enzyme-linked Immunosorbent Assay

    7) Product Images from "SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus"

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01649-6

    Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.
    Figure Legend Snippet: Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.

    Techniques Used: Neutralization, Incubation, Expressing, Flow Cytometry, Infection, Two Tailed Test, MANN-WHITNEY

    SARS-CoV-2 specific antibody detection assay. a Illustration of antibody detection assay. Biotinylated S-RBD or Nucleocapsid proteins are captured by streptavidin-coated beads, then incubated with plasma samples and stained with PE-conjugated anti-IgG, IgA, IgM, IgG1, IgG2, IgG3, IgG4 antibodies. Fluorescence intensity analyzed by flow cytometry. b Histogram overlays demonstrating the detection of anti-S-RBD human IgG antibody (left) and soluble ACE2-Fc (right) as positive controls for plasma antibody assay. c Representative patient plasma titration. Healthy control plasma at 1:100 dilution was used as a negative control. Serial dilutions were used in the flow cytometry overlay. d Comparison of IgG antibody levels captured by S-RBD, S1 subunit of spike, S1 N terminal domain (NTD), S2 extracellular domain (ECD) and nucleocapsid protein coated beads ( n = 46 biologically independent samples). e Correlation and comparison of bead-based assay S-RBD IgG antibody levels with ELISA-based assay ( n = 44). Two-tailed Mann–Whitney U test was used to determine the statistical significance in ( d ) and two-tailed Spearman’s was used for correlation significance in ( e ). Horizontal bars in ( d ) and ( e ) indicate mean values.
    Figure Legend Snippet: SARS-CoV-2 specific antibody detection assay. a Illustration of antibody detection assay. Biotinylated S-RBD or Nucleocapsid proteins are captured by streptavidin-coated beads, then incubated with plasma samples and stained with PE-conjugated anti-IgG, IgA, IgM, IgG1, IgG2, IgG3, IgG4 antibodies. Fluorescence intensity analyzed by flow cytometry. b Histogram overlays demonstrating the detection of anti-S-RBD human IgG antibody (left) and soluble ACE2-Fc (right) as positive controls for plasma antibody assay. c Representative patient plasma titration. Healthy control plasma at 1:100 dilution was used as a negative control. Serial dilutions were used in the flow cytometry overlay. d Comparison of IgG antibody levels captured by S-RBD, S1 subunit of spike, S1 N terminal domain (NTD), S2 extracellular domain (ECD) and nucleocapsid protein coated beads ( n = 46 biologically independent samples). e Correlation and comparison of bead-based assay S-RBD IgG antibody levels with ELISA-based assay ( n = 44). Two-tailed Mann–Whitney U test was used to determine the statistical significance in ( d ) and two-tailed Spearman’s was used for correlation significance in ( e ). Horizontal bars in ( d ) and ( e ) indicate mean values.

    Techniques Used: Detection Assay, Incubation, Staining, Fluorescence, Flow Cytometry, Titration, Negative Control, Bead-based Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

    SARS-CoV-2 specific antibody detection in COVID-19 and convalescent plasma samples. a Measurement of spike protein and nucleocapsid protein-specific IgG and spike protein-specific IgM and IgA antibodies as described in Fig. 1 . Area under the curve (AUC) values of plasma antibodies were calculated from reciprocal dilution curves in antibody detection assay ( n = 256 for S-RBD IgG and Nucleocapsid IgG, n = 50 for S-RBD IgM, n = 144 for S-RBD IgA). Dotted lines indicate the negative threshold calculated by adding 1 standard deviation to the mean AUC values of healthy controls’ plasma. Horizontal bars show the mean value. Green, blue, salmon, red and yellow dots indicate negative controls, outpatient, hospitalized, ICU/deceased and plasma donor subjects, respectively. b S-RBD-specific IgG subclass AUC levels ( n = 144 for S-RBD IgG1, n = 74 for S-RBD IgG2, S-RBD IgG3 and S-RBD IgG4) c S-RBD IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115) d Nucleocapsid protein IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and convalescent plasma donors ( n = 115). e S-RBD IgA AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115). f S-RBD IgG AUC values of severity groups and plasma donors subdivided into males and females ( n = 115). Green dots show female subjects while purple squares indicate male subjects. Statistical significances were determined using two-tailed Mann–Whitney U test.
    Figure Legend Snippet: SARS-CoV-2 specific antibody detection in COVID-19 and convalescent plasma samples. a Measurement of spike protein and nucleocapsid protein-specific IgG and spike protein-specific IgM and IgA antibodies as described in Fig. 1 . Area under the curve (AUC) values of plasma antibodies were calculated from reciprocal dilution curves in antibody detection assay ( n = 256 for S-RBD IgG and Nucleocapsid IgG, n = 50 for S-RBD IgM, n = 144 for S-RBD IgA). Dotted lines indicate the negative threshold calculated by adding 1 standard deviation to the mean AUC values of healthy controls’ plasma. Horizontal bars show the mean value. Green, blue, salmon, red and yellow dots indicate negative controls, outpatient, hospitalized, ICU/deceased and plasma donor subjects, respectively. b S-RBD-specific IgG subclass AUC levels ( n = 144 for S-RBD IgG1, n = 74 for S-RBD IgG2, S-RBD IgG3 and S-RBD IgG4) c S-RBD IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115) d Nucleocapsid protein IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and convalescent plasma donors ( n = 115). e S-RBD IgA AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115). f S-RBD IgG AUC values of severity groups and plasma donors subdivided into males and females ( n = 115). Green dots show female subjects while purple squares indicate male subjects. Statistical significances were determined using two-tailed Mann–Whitney U test.

    Techniques Used: Detection Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY

    Neutralization of SARS-CoV-2 and SARS-CoV pseudoviruses with soluble ACE2 and Nabs. a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.
    Figure Legend Snippet: Neutralization of SARS-CoV-2 and SARS-CoV pseudoviruses with soluble ACE2 and Nabs. a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.

    Techniques Used: Neutralization, Incubation, Expressing, Infection, Blocking Assay, Standard Deviation

    Development of SARS-CoV-2 and SARS-CoV spike-protein pseudotyped lentiviruses. a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.
    Figure Legend Snippet: Development of SARS-CoV-2 and SARS-CoV spike-protein pseudotyped lentiviruses. a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.

    Techniques Used: Expressing, Staining, Transfection, Negative Control, Flow Cytometry, Infection, Plasmid Preparation, Generated, Incubation, Titration, Standard Deviation

    8) Product Images from "Testing of the inhibitory effects of loratadine and desloratadine on SARS-CoV-2 spike pseudotyped virus viropexis"

    Article Title: Testing of the inhibitory effects of loratadine and desloratadine on SARS-CoV-2 spike pseudotyped virus viropexis

    Journal: Chemico-Biological Interactions

    doi: 10.1016/j.cbi.2021.109420

    The effect of LOR and DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (A) The entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells after treated with 20 μM LOR and DES. (B) The effect of different concentration of DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (C) The fluorescence image of SARS-CoV-2 Spike pseudotyped virus infected ACE2 h cells in 24 h and 48 h after treated with 20 μM DES. Data are presented as mean ± S.D. n = 3, *p
    Figure Legend Snippet: The effect of LOR and DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (A) The entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells after treated with 20 μM LOR and DES. (B) The effect of different concentration of DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (C) The fluorescence image of SARS-CoV-2 Spike pseudotyped virus infected ACE2 h cells in 24 h and 48 h after treated with 20 μM DES. Data are presented as mean ± S.D. n = 3, *p

    Techniques Used: Concentration Assay, Fluorescence, Infection

    The molecular docking of LOR and DES with ACE2 receptor. (A) The chemical structural of LOR and DES. (B) The docking mode of ACE2 and SARS-CoV-2 spike protein, in which the contacting residues are shown as sticks at the SARS-CoV-2 RBD–ACE2 interfaces. (C) The docking mode of LOR with ACE2. (D) The docking mode of DES withACE2. (E) The docking mode of LOR and DES with ACE2 represented in cavity depth maps. ACE2 is shown in green, and the SARS-CoV-2 RBD core is shown in orange. DES is shown in blue, and LOR shown in purple. (PDB ID: 6M0J ).
    Figure Legend Snippet: The molecular docking of LOR and DES with ACE2 receptor. (A) The chemical structural of LOR and DES. (B) The docking mode of ACE2 and SARS-CoV-2 spike protein, in which the contacting residues are shown as sticks at the SARS-CoV-2 RBD–ACE2 interfaces. (C) The docking mode of LOR with ACE2. (D) The docking mode of DES withACE2. (E) The docking mode of LOR and DES with ACE2 represented in cavity depth maps. ACE2 is shown in green, and the SARS-CoV-2 RBD core is shown in orange. DES is shown in blue, and LOR shown in purple. (PDB ID: 6M0J ).

    Techniques Used:

    9) Product Images from "FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein"

    Article Title: FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein

    Journal: New Biotechnology

    doi: 10.1016/j.nbt.2021.01.010

    Isolation of four monobodies that bind the RBD of the SARS-CoV-2 virus by phage-display. (a) The 3D visualization of the fibronectin type III (FN3) domain (PDB: 1TTG) as shown in PyMOL, with the BC, DE, and FG loops labelled in different colors [ 66 ]. (b) Virions displaying the four monobody sequences (A, B, C, and D) were confirmed by ELISA to bind the RBD-Fc fusion protein and not to the Fc (negative control). Error bars represent standard error (SE) of triplicate measurements. (c) The amino acid sequences of the BC, DE, and FG loops within the four monobodies. Frequency represents the number of times a given monobody was identified among 9 confirmed binders. The complete primary structures of the four monobodies are shown in Suppl. Figure S3.
    Figure Legend Snippet: Isolation of four monobodies that bind the RBD of the SARS-CoV-2 virus by phage-display. (a) The 3D visualization of the fibronectin type III (FN3) domain (PDB: 1TTG) as shown in PyMOL, with the BC, DE, and FG loops labelled in different colors [ 66 ]. (b) Virions displaying the four monobody sequences (A, B, C, and D) were confirmed by ELISA to bind the RBD-Fc fusion protein and not to the Fc (negative control). Error bars represent standard error (SE) of triplicate measurements. (c) The amino acid sequences of the BC, DE, and FG loops within the four monobodies. Frequency represents the number of times a given monobody was identified among 9 confirmed binders. The complete primary structures of the four monobodies are shown in Suppl. Figure S3.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Negative Control

    Specificity of anti-RBD monobodies. The four MBP-FN3 fusions were adsorbed on microtiter plate wells and incubated with chemically biotinylated SARS-CoV-1 and SARS-CoV-2 RBD proteins mixed with a bacterial cell lysate. Wells coated with MBP alone served as a negative control and wells coated with an anti-spike monoclonal antibody, clone CR3022 [ 35 ], which binds equally well to the RBDs of both SARS-CoV-1 and SARS-CoV-2, served as a positive control. Binding of SARS-CoV-1 and SARS-CoV-2 RBD-Fc fusion proteins was revealed with streptavidin-HRP. Error bars represent standard error (SE) of triplicate measurements.
    Figure Legend Snippet: Specificity of anti-RBD monobodies. The four MBP-FN3 fusions were adsorbed on microtiter plate wells and incubated with chemically biotinylated SARS-CoV-1 and SARS-CoV-2 RBD proteins mixed with a bacterial cell lysate. Wells coated with MBP alone served as a negative control and wells coated with an anti-spike monoclonal antibody, clone CR3022 [ 35 ], which binds equally well to the RBDs of both SARS-CoV-1 and SARS-CoV-2, served as a positive control. Binding of SARS-CoV-1 and SARS-CoV-2 RBD-Fc fusion proteins was revealed with streptavidin-HRP. Error bars represent standard error (SE) of triplicate measurements.

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

    Purification of RBD-Fc, ACE2-Fc, and spike protein. (a) Recombinant. SARS-CoV-2 spike RBD-Fc fusion protein. The predicted molecular weight (MW) is ∼ 65 kDa, when resolved by SDS-PAGE under reducing conditions with sized standards (MW shown in kDa); > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (b) Recombinant ACE2-Fc fusion protein. The predicted MW is ∼110 kDa, when resolved by SDS-PAGE under reducing conditions, and judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (c) Spike protein. The near full-length protein resolved as a doublet with a MW of ∼170 kDa under reducing conditions and was judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. The doublet bands are thought to differ in post-translational modifications. Composite image of two lanes from the same gel.
    Figure Legend Snippet: Purification of RBD-Fc, ACE2-Fc, and spike protein. (a) Recombinant. SARS-CoV-2 spike RBD-Fc fusion protein. The predicted molecular weight (MW) is ∼ 65 kDa, when resolved by SDS-PAGE under reducing conditions with sized standards (MW shown in kDa); > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (b) Recombinant ACE2-Fc fusion protein. The predicted MW is ∼110 kDa, when resolved by SDS-PAGE under reducing conditions, and judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (c) Spike protein. The near full-length protein resolved as a doublet with a MW of ∼170 kDa under reducing conditions and was judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. The doublet bands are thought to differ in post-translational modifications. Composite image of two lanes from the same gel.

    Techniques Used: Purification, Recombinant, Molecular Weight, SDS Page, Staining

    Detection of the SARS-CoV-2 RBD in a complex biological mixture. An E. coli cell lysate was mixed with various concentrations of SARS-CoV-2 RBD and added to microtiter wells coated with the FN3A-MBP fusion protein. After incubation and washing of the wells, the ectodomain of ACE2, conjugated to HRP, was added. Negative controls consisted of MBP in lieu of FN3A and Fc alone in lieu of RBD. Error bars represent SE of triplicate measurements.
    Figure Legend Snippet: Detection of the SARS-CoV-2 RBD in a complex biological mixture. An E. coli cell lysate was mixed with various concentrations of SARS-CoV-2 RBD and added to microtiter wells coated with the FN3A-MBP fusion protein. After incubation and washing of the wells, the ectodomain of ACE2, conjugated to HRP, was added. Negative controls consisted of MBP in lieu of FN3A and Fc alone in lieu of RBD. Error bars represent SE of triplicate measurements.

    Techniques Used: Incubation

    10) Product Images from "Testing of the inhibitory effects of loratadine and desloratadine on SARS-CoV-2 spike pseudotyped virus viropexis"

    Article Title: Testing of the inhibitory effects of loratadine and desloratadine on SARS-CoV-2 spike pseudotyped virus viropexis

    Journal: Chemico-Biological Interactions

    doi: 10.1016/j.cbi.2021.109420

    The effect of LOR and DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (A) The entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells after treated with 20 μM LOR and DES. (B) The effect of different concentration of DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (C) The fluorescence image of SARS-CoV-2 Spike pseudotyped virus infected ACE2 h cells in 24 h and 48 h after treated with 20 μM DES. Data are presented as mean ± S.D. n = 3, *p
    Figure Legend Snippet: The effect of LOR and DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (A) The entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells after treated with 20 μM LOR and DES. (B) The effect of different concentration of DES on the entrance of SARS-CoV-2 Spike pseudotyped virus into ACE2 h cells. (C) The fluorescence image of SARS-CoV-2 Spike pseudotyped virus infected ACE2 h cells in 24 h and 48 h after treated with 20 μM DES. Data are presented as mean ± S.D. n = 3, *p

    Techniques Used: Concentration Assay, Fluorescence, Infection

    The molecular docking of LOR and DES with ACE2 receptor. (A) The chemical structural of LOR and DES. (B) The docking mode of ACE2 and SARS-CoV-2 spike protein, in which the contacting residues are shown as sticks at the SARS-CoV-2 RBD–ACE2 interfaces. (C) The docking mode of LOR with ACE2. (D) The docking mode of DES withACE2. (E) The docking mode of LOR and DES with ACE2 represented in cavity depth maps. ACE2 is shown in green, and the SARS-CoV-2 RBD core is shown in orange. DES is shown in blue, and LOR shown in purple. (PDB ID: 6M0J ).
    Figure Legend Snippet: The molecular docking of LOR and DES with ACE2 receptor. (A) The chemical structural of LOR and DES. (B) The docking mode of ACE2 and SARS-CoV-2 spike protein, in which the contacting residues are shown as sticks at the SARS-CoV-2 RBD–ACE2 interfaces. (C) The docking mode of LOR with ACE2. (D) The docking mode of DES withACE2. (E) The docking mode of LOR and DES with ACE2 represented in cavity depth maps. ACE2 is shown in green, and the SARS-CoV-2 RBD core is shown in orange. DES is shown in blue, and LOR shown in purple. (PDB ID: 6M0J ).

    Techniques Used:

    11) Product Images from "Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry"

    Article Title: Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

    Journal: bioRxiv

    doi: 10.1101/2021.02.28.433291

    High-resolution glycoform characterization of intact S-RBD by top-down MS. (A-D) Illustration of the top-down glycoproteomics workflow for the comprehensive analysis of the S-RBD glycoforms. (A) The SARS-CoV-2 coronavirus features a surface S protein that possesses a glycosylated RBD (highlighted in the dashed box in B). (B) Intact glycoprotein analysis proceeds by directly infusing solubilized S-RBD and electrospraying S-RBD protein ions into either (C) a hybrid trapped ion mobility spectrometry (TIMS) MS device (timsTOF) for ion mobility MS analysis, or (D) an ultrahigh-resolution Fourier transform ion cyclotron (FTICR) MS. (E) Specific isolation of S-RBD proteoforms by top-down MS analysis, illustrating raw MS 1 and corresponding deconvoluted protein spectrum, for structural characterizations of glycoforms. PDB: 6M0J.
    Figure Legend Snippet: High-resolution glycoform characterization of intact S-RBD by top-down MS. (A-D) Illustration of the top-down glycoproteomics workflow for the comprehensive analysis of the S-RBD glycoforms. (A) The SARS-CoV-2 coronavirus features a surface S protein that possesses a glycosylated RBD (highlighted in the dashed box in B). (B) Intact glycoprotein analysis proceeds by directly infusing solubilized S-RBD and electrospraying S-RBD protein ions into either (C) a hybrid trapped ion mobility spectrometry (TIMS) MS device (timsTOF) for ion mobility MS analysis, or (D) an ultrahigh-resolution Fourier transform ion cyclotron (FTICR) MS. (E) Specific isolation of S-RBD proteoforms by top-down MS analysis, illustrating raw MS 1 and corresponding deconvoluted protein spectrum, for structural characterizations of glycoforms. PDB: 6M0J.

    Techniques Used: Ion-Mobility Spectrometry, Isolation

    Related Articles

    Incubation:

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry
    Article Snippet: Cells were collected with TrypLE (Thermo #12605010) and washed twice with ice-cold PBS. .. Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C. .. After two additional washes, cells were subjected to flow cytometry analysis (Thermo, Attune™ NxT) and data processing (FlowJo v10.0.7).

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants
    Article Snippet: The SARS-CoV-2-specific IgG antibody titer was measured by ELISA. .. Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates. .. Finally, the absorbance (450/630 nm) was measured with Infinite M200 (TECAN).

    Recombinant:

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry
    Article Snippet: Cells were collected with TrypLE (Thermo #12605010) and washed twice with ice-cold PBS. .. Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C. .. After two additional washes, cells were subjected to flow cytometry analysis (Thermo, Attune™ NxT) and data processing (FlowJo v10.0.7).

    Article Title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19
    Article Snippet: The PRNT50 and PRNT90 titers were calculated as the last serum dilution resulting in at least 50% and 90% SARS-CoV-2 neutralization, respectively. .. Proteins Recombinant SARS-CoV-2 spike receptor binding domain (RBD) and its mutants were purchased from Sino Biologicals (RBD-wt; 40592-V08H82, RBD-K417N; 40592-V08H59, RBD-N501Y; 40592-V08H82 and RBD-E484K; 40592-V08H84). .. Recombinant purified RBD proteins used in the study were produced in 293 mammalian cells.

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants
    Article Snippet: The SARS-CoV-2-specific IgG antibody titer was measured by ELISA. .. Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates. .. Finally, the absorbance (450/630 nm) was measured with Infinite M200 (TECAN).

    Staining:

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry
    Article Snippet: Cells were collected with TrypLE (Thermo #12605010) and washed twice with ice-cold PBS. .. Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C. .. After two additional washes, cells were subjected to flow cytometry analysis (Thermo, Attune™ NxT) and data processing (FlowJo v10.0.7).

    other:

    Article Title: Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity
    Article Snippet: The S2 ectodomain of the SARS-CoV-2 spike protein (aa 686–1213) was purchased from Sino Biological, Eschborn, Germany (cat # 40590, lot # LC14MC3007).

    Binding Assay:

    Article Title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19
    Article Snippet: The PRNT50 and PRNT90 titers were calculated as the last serum dilution resulting in at least 50% and 90% SARS-CoV-2 neutralization, respectively. .. Proteins Recombinant SARS-CoV-2 spike receptor binding domain (RBD) and its mutants were purchased from Sino Biologicals (RBD-wt; 40592-V08H82, RBD-K417N; 40592-V08H59, RBD-N501Y; 40592-V08H82 and RBD-E484K; 40592-V08H84). .. Recombinant purified RBD proteins used in the study were produced in 293 mammalian cells.

    Expressing:

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus
    Article Snippet: Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (Tree Star). .. Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol. .. Viral supernatants were collected 24–48 h post-transfection, filtered through a 0.45 μm syringe filter (Millipore) to remove cellular debris, and concentrated with Lenti-X (Invitrogen) according to the manufacturers' protocol.

    Plasmid Preparation:

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus
    Article Snippet: Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (Tree Star). .. Pseudotyped lentivirus production and titer measurement Lentivector plasmids containing RFP or GFP reporter genes were co-transfected with either SARS-CoV-2 spike protein or SARS -CoV spike protein (Human SARS coronavirus (SARS-CoV) spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized) from SinoBiological) plasmids into HEK-293T cells using Lipofectamine TM 3000 (Invitrogen) according to the manufacturer’s protocol. .. Viral supernatants were collected 24–48 h post-transfection, filtered through a 0.45 μm syringe filter (Millipore) to remove cellular debris, and concentrated with Lenti-X (Invitrogen) according to the manufacturers' protocol.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants
    Article Snippet: The SARS-CoV-2-specific IgG antibody titer was measured by ELISA. .. Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates. .. Finally, the absorbance (450/630 nm) was measured with Infinite M200 (TECAN).

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    Sino Biological sars cov 2 spike
    Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of <t>SARS-CoV-2,</t> and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR
    Sars Cov 2 Spike, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of SARS-CoV-2, and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR

    Journal: Nature Communications

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

    doi: 10.1038/s41467-021-21213-4

    Figure Lengend Snippet: Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of SARS-CoV-2, and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR

    Article Snippet: Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C.

    Techniques: Genome Wide, CRISPR, Expressing, Knock-Out, Infection, Sequencing

    Host genes that regulate the surface expression of receptor ACE2 are identified. a The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37 °C for internalization. Viral RNA was extracted for RT-qPCR analysis (two experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). b , c Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody (2 experiments; n = 7 ( b ) or 6 ( c ); one-way ANOVA with Dunnett’s test; mean ± s.d.). d , e Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for western blotting. One representative blot was shown ( d ) and data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments ( e ) (four experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). f , g The impact on viral production in CCDC53 gene-edited Calu-3 cells. The mixed cell population was infected with Sfull ( f ) or Sdel ( g ) to assess the virus yield (two experiments; n = 6; two-way ANOVA with Sidak’s test). * P

    Journal: Nature Communications

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

    doi: 10.1038/s41467-021-21213-4

    Figure Lengend Snippet: Host genes that regulate the surface expression of receptor ACE2 are identified. a The effect on virion binding and internalization in gene-edited cells. A549-ACE2 cells were incubated with SARS-CoV-2 Sfull infectious virus on ice for binding or then switched to 37 °C for internalization. Viral RNA was extracted for RT-qPCR analysis (two experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). b , c Surface expression of receptor ACE2 was decreased in gene-edited cells as measured by flow cytometry using S1-Fc recombinant protein or anti-ACE2 antibody (2 experiments; n = 7 ( b ) or 6 ( c ); one-way ANOVA with Dunnett’s test; mean ± s.d.). d , e Surface and total expression of receptor ACE2 were decreased in gene-edited cells. The plasma membrane proteins were biotin-labeled and immunoprecipitated by streptavidin beads for western blotting. One representative blot was shown ( d ) and data are pooled from four independent experiments, quantified, and normalized to the controls of individual experiments ( e ) (four experiments; n = 4; one-way ANOVA with Dunnett’s test; mean ± s.d.). f , g The impact on viral production in CCDC53 gene-edited Calu-3 cells. The mixed cell population was infected with Sfull ( f ) or Sdel ( g ) to assess the virus yield (two experiments; n = 6; two-way ANOVA with Sidak’s test). * P

    Article Snippet: Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C.

    Techniques: Expressing, Binding Assay, Incubation, Quantitative RT-PCR, Flow Cytometry, Recombinant, Labeling, Immunoprecipitation, Western Blot, Infection

    Genes identified are required for the endosomal cell entry of SARS-CoV-2, SARS-CoV, and MERS-CoV. a–d The selected genes were verified for the infection by pseudovirus bearing the spike protein of SARS-CoV-2 Sdel strain ( a ), the the glycoprotein of vesicular stomatitis virus (VSV-G) ( b ), the spike protein of SARS-CoV ( c ), or the spike protein of MERS-CoV ( d ) (two experiments; n = 4–11; one-way ANOVA with Dunnett’s test; mean ± s.d.). One representative sgRNA per gene was used in A549-ACE2 cells. e The genes selected were verified for the infection by the SARS-CoV-2 Sfull live virus (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). f Effect of NPC1 inhibitor U18666A on virus infection. Cells were treated with U18666A at the indicated concentrations 2 h prior to or 2 h post infection by Sfull or Sdel live virus. The viral N-positive cells were calculated (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the controls of individual experiments. ** P

    Journal: Nature Communications

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

    doi: 10.1038/s41467-021-21213-4

    Figure Lengend Snippet: Genes identified are required for the endosomal cell entry of SARS-CoV-2, SARS-CoV, and MERS-CoV. a–d The selected genes were verified for the infection by pseudovirus bearing the spike protein of SARS-CoV-2 Sdel strain ( a ), the the glycoprotein of vesicular stomatitis virus (VSV-G) ( b ), the spike protein of SARS-CoV ( c ), or the spike protein of MERS-CoV ( d ) (two experiments; n = 4–11; one-way ANOVA with Dunnett’s test; mean ± s.d.). One representative sgRNA per gene was used in A549-ACE2 cells. e The genes selected were verified for the infection by the SARS-CoV-2 Sfull live virus (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). f Effect of NPC1 inhibitor U18666A on virus infection. Cells were treated with U18666A at the indicated concentrations 2 h prior to or 2 h post infection by Sfull or Sdel live virus. The viral N-positive cells were calculated (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the controls of individual experiments. ** P

    Article Snippet: Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C.

    Techniques: Infection

    The deletion at the S1/S2 boundary of spike protein propels the virus to enter cells through the endosomal pathway. a Sequence alignment of spike protein encompassing the cleavage site between S1 and S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel strain) deletion were used to compare with that of SARS-CoV. The insertion of multi-basic amino acids in spike protein of SARS-CoV-2 was shown in red. b Comparison of the replication property between Sfull and Sdel strains in different cell lines. The percentage of nucleocapsid (N) protein-positive cells was analyzed by imaging-based analysis following virus infection (two or more experiments; n = 6 except for Calu-3 in which n = 8; one-way ANOVA with Dunnett’s test; mean ± s.d.). c Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data are normalized to the Sfull of individual experiments (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). d Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines (two experiments; n = 4 or 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the untreated group of individual experiments. **** P

    Journal: Nature Communications

    Article Title: A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

    doi: 10.1038/s41467-021-21213-4

    Figure Lengend Snippet: The deletion at the S1/S2 boundary of spike protein propels the virus to enter cells through the endosomal pathway. a Sequence alignment of spike protein encompassing the cleavage site between S1 and S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel strain) deletion were used to compare with that of SARS-CoV. The insertion of multi-basic amino acids in spike protein of SARS-CoV-2 was shown in red. b Comparison of the replication property between Sfull and Sdel strains in different cell lines. The percentage of nucleocapsid (N) protein-positive cells was analyzed by imaging-based analysis following virus infection (two or more experiments; n = 6 except for Calu-3 in which n = 8; one-way ANOVA with Dunnett’s test; mean ± s.d.). c Evaluation of entry efficiency in different cell lines infected with pseudoviruses bearing spike protein Sfull, Sdel, or S mutant (R682S, R685S). Data are normalized to the Sfull of individual experiments (two experiments; n = 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). d Effect of TMPRSS2 serine protease inhibitor Camostat and cysteine protease inhibitor E-64d on Sfull or Sdel infection in different cell lines (two experiments; n = 4 or 6; one-way ANOVA with Dunnett’s test; mean ± s.d.). Data shown were normalized to the untreated group of individual experiments. **** P

    Article Snippet: Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1 μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min. After washing, cells were stained with goat anti-human IgG (H + L) conjugated with Alexa Fluor 647 (Thermo #A21445, 2 μg/ml) for 30 min at 4 °C.

    Techniques: Sequencing, Imaging, Infection, Mutagenesis, Protease Inhibitor

    SARS-CoV-2 specific T cell immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD4 + T cells. ( B ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD4 + effector memory T cells (CD44 + CD62L - ) in splenocytes. ( C ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD8 + T. ( D ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD8 + effector memory T cells (CD44 + CD62L - ) in splenocytes. Results were pooled from two independent experiments ( B and D ). Data are presented as the mean ± S.E.M. in C and D, n = 3 or 4 for each group. Each symbol represents an individual mouse.

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: SARS-CoV-2 specific T cell immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD4 + T cells. ( B ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD4 + effector memory T cells (CD44 + CD62L - ) in splenocytes. ( C ) The FACS analysis results showing the percentages of cytokine positive cells evaluated among single and viable CD44 + CD62L - CD8 + T. ( D ) The intracellular staining assay for cytokines (IFN-γ, TNF-α, and IL-2) production among SARS-CoV-2 specific CD8 + effector memory T cells (CD44 + CD62L - ) in splenocytes. Results were pooled from two independent experiments ( B and D ). Data are presented as the mean ± S.E.M. in C and D, n = 3 or 4 for each group. Each symbol represents an individual mouse.

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: Mouse Assay, FACS, Staining

    The FACS chromatogram of the competitive inhibition of SARS-CoV-2 pseudovirus infection (harboring EGFP reporter) by the circRNA RBD -translated RBD antigens.

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: The FACS chromatogram of the competitive inhibition of SARS-CoV-2 pseudovirus infection (harboring EGFP reporter) by the circRNA RBD -translated RBD antigens.

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: FACS, Inhibition, Infection

    The susceptibility of SARS-CoV-2 D614G, B.1.1.7 or B.1.351variants to neutralizing antibodies elicited by the circRNA RBD or circRNA RBD-501Y.V2 vaccines in mice. ( A ) Schematic diagram of circRNA RBD-501Y.V2 circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD-501Y.V2, the RBD antigen harboring the K417N-E484K-N501Y mutations in SARS-CoV-2 501Y.V2 variant. ( B ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data was shown as the mean ± S.E.M. Each symbol represents an individual mouse. ( C ) Sigmodal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD-501Y.V2 (50 μg) immunized mice were collected at 1 week or 2 weeks post boost. The data were shown as the mean ± S.E.M. ( D ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD vaccines. The serum samples were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( E ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD-501Y.V2 vaccines. The serum samples were collected at 1 week post boost. The data were shown as the mean ± S.E.M. (n = 5).

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: The susceptibility of SARS-CoV-2 D614G, B.1.1.7 or B.1.351variants to neutralizing antibodies elicited by the circRNA RBD or circRNA RBD-501Y.V2 vaccines in mice. ( A ) Schematic diagram of circRNA RBD-501Y.V2 circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD-501Y.V2, the RBD antigen harboring the K417N-E484K-N501Y mutations in SARS-CoV-2 501Y.V2 variant. ( B ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data was shown as the mean ± S.E.M. Each symbol represents an individual mouse. ( C ) Sigmodal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD-501Y.V2 (50 μg) immunized mice were collected at 1 week or 2 weeks post boost. The data were shown as the mean ± S.E.M. ( D ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD vaccines. The serum samples were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( E ) Neutralization assay of VSV-based D614G, B.1.1.7 or B.1.351 pseudovirus with the serum of mice immunized with circRNA RBD-501Y.V2 vaccines. The serum samples were collected at 1 week post boost. The data were shown as the mean ± S.E.M. (n = 5).

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: Mouse Assay, Sequencing, Variant Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Neutralization

    Expression of SARS-CoV-2 neutralizing nanobodies or hACE2 decoys via circRNA platform. ( A ) Schematic diagram of circRNA nAB or circRNA hACE2 decoys circularization by the Group I ribozyme autocatalysis. ( B ) Lentivirial-based pseudovirus neutralization assay with the supernatant from cells transfected with circRNA encoding neutralizing nanobodies nAB1, nAB1-Tri, nAB2, nAB2-Tri, nAB3 and nAB3-Tri or ACE2 decoys. The luciferase value was normalized to the circRNA EGFP control. The data was shown as the mean ± S.E.M. (n = 2).

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: Expression of SARS-CoV-2 neutralizing nanobodies or hACE2 decoys via circRNA platform. ( A ) Schematic diagram of circRNA nAB or circRNA hACE2 decoys circularization by the Group I ribozyme autocatalysis. ( B ) Lentivirial-based pseudovirus neutralization assay with the supernatant from cells transfected with circRNA encoding neutralizing nanobodies nAB1, nAB1-Tri, nAB2, nAB2-Tri, nAB3 and nAB3-Tri or ACE2 decoys. The luciferase value was normalized to the circRNA EGFP control. The data was shown as the mean ± S.E.M. (n = 2).

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: Expressing, Neutralization, Transfection, Luciferase

    Humoral immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) Schematic representation of LNP-circRNA complex. ( B ) Representative of concentration-size graph of LNP-circRNA RBD measured by dynamic light scattering method. ( C ) Schematic diagram of the LNP-circRNA RBD vaccination process in BALB/c mice and serum collection schedule for specific antibodies analysis. ( D ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data were shown as the mean ± S.E.M. (n = 4 or 5). ( E ) Sigmoidal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 2 weeks post the second dose. The data was shown as the mean ± S.E.M. (n = 4). ( F ) Sigmoldal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( G ) The NT50 was calculated using lentivirus-based SARS-CoV-2 pseudovirus. The data was shown as the mean ± S.E.M. (n = 5).

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: Humoral immune responses in mice immunized with SARS-CoV-2 circRNA RBD vaccines. ( A ) Schematic representation of LNP-circRNA complex. ( B ) Representative of concentration-size graph of LNP-circRNA RBD measured by dynamic light scattering method. ( C ) Schematic diagram of the LNP-circRNA RBD vaccination process in BALB/c mice and serum collection schedule for specific antibodies analysis. ( D ) Measuring the SARS-CoV-2 specific IgG antibody titer with ELISA. The data were shown as the mean ± S.E.M. (n = 4 or 5). ( E ) Sigmoidal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 2 weeks post the second dose. The data was shown as the mean ± S.E.M. (n = 4). ( F ) Sigmoldal curve diagram of the inhibition rate by sera of immunized mice with surrogate virus neutralization assay. Sera from circRNA RBD (10 μg) and circRNA RBD (50 μg) immunized mice were collected at 5 weeks post boost. The data were shown as the mean ± S.E.M. (n = 5). ( G ) The NT50 was calculated using lentivirus-based SARS-CoV-2 pseudovirus. The data was shown as the mean ± S.E.M. (n = 5).

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

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

    Flow panel and gating strategy to quantify SARS-CoV-2-RBD-specific T cells in mice. The plots showed the gating strategy of single and viable T cells from spleens. CD4 + or CD8 + T cells were further analyzed with the expression of CD44 and CD62L.

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: Flow panel and gating strategy to quantify SARS-CoV-2-RBD-specific T cells in mice. The plots showed the gating strategy of single and viable T cells from spleens. CD4 + or CD8 + T cells were further analyzed with the expression of CD44 and CD62L.

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: Mouse Assay, Expressing

    Expression of trimeric SARS-CoV-2 RBD antigens with circular RNAs in vitro . ( A ) Schematic diagram of circRNA RBD circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD, the receptor binding domain of SARS-CoV-2 Spike protein. The arrows indicate the the design of primers for PCR analysis. ( B ) The agarose gel electrophoresis result of the PCR products of linear RNA RBD and circRNA RBD . ( C ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD . The circRNA EGFP and linear RNA RBD were set as controls. ( D ) The quantitative ELISA assay to measure the concentration of RBD antigens in the supernatant. The data in ( B ) was shown as the mean ± S.E.M. (n = 3). ( E ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of mouse NIH3T3 cells transfected with circRNA RBD . The circRNA EGFP was set as controls. ( F ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD for different shelf time (3, 7 or 14 days) at room temperature (∼25°C). ( G ) Quantification of the competitive inhibition of SARS-CoV-2 pseudovirus infection (EGFP) by the circRNA RBD -translated RBD antigens. The circRNA EGFP and linear RNA RBD were set as controls. The data in ( E ) was shown as the mean ± S.E.M. (n = 2).

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: Expression of trimeric SARS-CoV-2 RBD antigens with circular RNAs in vitro . ( A ) Schematic diagram of circRNA RBD circularization by the Group I ribozyme autocatalysis. SP, signal peptide sequence of human tPA protein. T4, the trimerization domain from bacteriophage T4 fibritin protein. RBD, the receptor binding domain of SARS-CoV-2 Spike protein. The arrows indicate the the design of primers for PCR analysis. ( B ) The agarose gel electrophoresis result of the PCR products of linear RNA RBD and circRNA RBD . ( C ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD . The circRNA EGFP and linear RNA RBD were set as controls. ( D ) The quantitative ELISA assay to measure the concentration of RBD antigens in the supernatant. The data in ( B ) was shown as the mean ± S.E.M. (n = 3). ( E ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of mouse NIH3T3 cells transfected with circRNA RBD . The circRNA EGFP was set as controls. ( F ) Western Blot analysis showing the expression level of RBD antigens in the supernatant of HEK293T cells transfected with circRNA RBD for different shelf time (3, 7 or 14 days) at room temperature (∼25°C). ( G ) Quantification of the competitive inhibition of SARS-CoV-2 pseudovirus infection (EGFP) by the circRNA RBD -translated RBD antigens. The circRNA EGFP and linear RNA RBD were set as controls. The data in ( E ) was shown as the mean ± S.E.M. (n = 2).

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: Expressing, In Vitro, Sequencing, Binding Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition, Infection

    Identification of IL-4 producing CD4 + T cells in mice immunized with SARS-CoV-2 circRNA RBD vaccines. Splenocytes were stimulated with SARS-CoV-2-RBD peptides for 7 hr in the presence of BFA and Monensin. PMA and Ionomycin stimulation were applied as a positive control. Cells were gated on single and viable CD4 + T cells. The plots are representative for two independent experiments with same results.

    Journal: bioRxiv

    Article Title: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants

    doi: 10.1101/2021.03.16.435594

    Figure Lengend Snippet: Identification of IL-4 producing CD4 + T cells in mice immunized with SARS-CoV-2 circRNA RBD vaccines. Splenocytes were stimulated with SARS-CoV-2-RBD peptides for 7 hr in the presence of BFA and Monensin. PMA and Ionomycin stimulation were applied as a positive control. Cells were gated on single and viable CD4 + T cells. The plots are representative for two independent experiments with same results.

    Article Snippet: Briefly, serial 3-fold dilutions (in 1% BSA) of heat-inactivated sera, starting at 1:50, were added to the 96-well plates (100 μL/well; Costar) coated with recombinant SARS-CoV-2 Spike antigens (Sino Biological) and blocked with 1% BSA, and the plates were incubated for at 37°C for 60 min. Then, after three washes with wash buffer, the Horseradish peroxidase HRP-conjugated rabbit anti-mouse IgG (Sigma) diluted in 1% BSA at 1:10,000 ratio (Sigma), was added to the plates and incubated at 37°C for 45 min. Then the plates were washed for 4 times with wash buffer and added with TMB substrates (100 μL/well) followed by incubation for 15-20 min. And then the ELISA stop buffer was added into the plates.

    Techniques: Mouse Assay, Positive Control

    Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

    Journal: bioRxiv

    Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

    doi: 10.1101/2021.03.01.433404

    Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

    Article Snippet: Briefly, a monoclonal antibody directed against the spike protein of SARS-CoV-2 (1:1,500, Sino Biological) was detected with a peroxidase-conjugated anti-rabbit secondary antibody (1:1,000, Dianova), followed by addition of AEC substrate.

    Techniques: Derivative Assay, Infection

    SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from [ Bojkova et al., 2020 ]. P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from [ Blanco-Melo et al., 2020 ]). P-values were determined by two-sided Student’s t-test.

    Journal: bioRxiv

    Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

    doi: 10.1101/2021.03.01.433404

    Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from [ Bojkova et al., 2020 ]. P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from [ Blanco-Melo et al., 2020 ]). P-values were determined by two-sided Student’s t-test.

    Article Snippet: Briefly, a monoclonal antibody directed against the spike protein of SARS-CoV-2 (1:1,500, Sino Biological) was detected with a peroxidase-conjugated anti-rabbit secondary antibody (1:1,000, Dianova), followed by addition of AEC substrate.

    Techniques: Infection, Derivative Assay, Polymerase Chain Reaction

    Neutralizing antibody titers and RBD binding antibodies of convalescent plasma and hCoV-2IG against various SARS-CoV-2 strains. (A) SARS-CoV-2 neutralizing antibody titers in CP, 2019-IVIG and hCoV-2IG preparations as determined by pseudovirus neutralization assay in 293-ACE2-TMPRSS2 cells with SARS-CoV-2 WA-1 strain, CA variant (B.1.429), UK variant (B.1.1.7), JP variant (P.1) or SA variant (B.1.351). PsVNA50 (50% neutralization titer) and PsVNA80 (80% neutralization titer) titers for control pre-pandemic 2019-IVIG (n=16), convalescent plasma (n =9) and hCoV-2IG (n = 6) were calculated with GraphPad prism version 8. Data show mean values + SEM for PsVNA50 and PsVNA80 titers for each of the 3 antibody groups against the SARS-CoV-2 WA-1, CA, UK, JP and SA variants. (B) End-point virus neutralization titers for six hCoV-2IG lots using wild type authentic SARS-CoV-2 WA-1, UK and SA virus strains in a classical BSL3 neutralization assay based on a plaque assay was performed as described in Materials and Methods. (C) Pearson two-tailed correlations are reported for the calculation of correlation of PRNT50 titers against wild-type SARS-CoV-2 strains (WA-1, UK or SA) and PsVNA50 titers against corresponding pseudovirions expressing either WA-1, UK or SA spike in pseudovirion neutralization assays for the six hCOV-2IG lots. (D) Antibody concentration (in mg/mL) required for each of the six hCoV-2IG batches to achieve 50% neutralization of SARS-CoV-2 WA-1, CA, UK, JP or SA variants in PsVNA. (E-F) Fold-decrease in PsVNA50 neutralization titers against emerging variant strain CA (B.1.429), UK (B.1.1.7), JP (P.1) and SA (B.1.351) for six hCoV-2IG lots (E) and nine CP lots (F) in comparison with SARS-CoV-2 WA-1 strain. The numbers above the group shows the mean fold-change for each variant. (G-H) Total antibody binding (Max RU) of 1mg/mL for the six batches of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6) to purified WA-1 RBD (RBD-wt) and RBD mutants: RBD-K417N, RBD-N501Y and RBD-E484K by SPR (G). The numbers above the group show the mean antibody binding for each RBD. (H) Fold-decrease in antibody binding to mutants RBD-K417N, RBD-N501Y and RBD-E484K of hCoV-2IG in comparison with RBD-wt from WA-1 strain calculated from the data in Panel G. The numbers above the group shows the mean fold-change for each mutant RBD. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Journal: bioRxiv

    Article Title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

    doi: 10.1101/2021.03.19.436183

    Figure Lengend Snippet: Neutralizing antibody titers and RBD binding antibodies of convalescent plasma and hCoV-2IG against various SARS-CoV-2 strains. (A) SARS-CoV-2 neutralizing antibody titers in CP, 2019-IVIG and hCoV-2IG preparations as determined by pseudovirus neutralization assay in 293-ACE2-TMPRSS2 cells with SARS-CoV-2 WA-1 strain, CA variant (B.1.429), UK variant (B.1.1.7), JP variant (P.1) or SA variant (B.1.351). PsVNA50 (50% neutralization titer) and PsVNA80 (80% neutralization titer) titers for control pre-pandemic 2019-IVIG (n=16), convalescent plasma (n =9) and hCoV-2IG (n = 6) were calculated with GraphPad prism version 8. Data show mean values + SEM for PsVNA50 and PsVNA80 titers for each of the 3 antibody groups against the SARS-CoV-2 WA-1, CA, UK, JP and SA variants. (B) End-point virus neutralization titers for six hCoV-2IG lots using wild type authentic SARS-CoV-2 WA-1, UK and SA virus strains in a classical BSL3 neutralization assay based on a plaque assay was performed as described in Materials and Methods. (C) Pearson two-tailed correlations are reported for the calculation of correlation of PRNT50 titers against wild-type SARS-CoV-2 strains (WA-1, UK or SA) and PsVNA50 titers against corresponding pseudovirions expressing either WA-1, UK or SA spike in pseudovirion neutralization assays for the six hCOV-2IG lots. (D) Antibody concentration (in mg/mL) required for each of the six hCoV-2IG batches to achieve 50% neutralization of SARS-CoV-2 WA-1, CA, UK, JP or SA variants in PsVNA. (E-F) Fold-decrease in PsVNA50 neutralization titers against emerging variant strain CA (B.1.429), UK (B.1.1.7), JP (P.1) and SA (B.1.351) for six hCoV-2IG lots (E) and nine CP lots (F) in comparison with SARS-CoV-2 WA-1 strain. The numbers above the group shows the mean fold-change for each variant. (G-H) Total antibody binding (Max RU) of 1mg/mL for the six batches of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6) to purified WA-1 RBD (RBD-wt) and RBD mutants: RBD-K417N, RBD-N501Y and RBD-E484K by SPR (G). The numbers above the group show the mean antibody binding for each RBD. (H) Fold-decrease in antibody binding to mutants RBD-K417N, RBD-N501Y and RBD-E484K of hCoV-2IG in comparison with RBD-wt from WA-1 strain calculated from the data in Panel G. The numbers above the group shows the mean fold-change for each mutant RBD. All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Article Snippet: Proteins Recombinant SARS-CoV-2 spike receptor binding domain (RBD) and its mutants were purchased from Sino Biologicals (RBD-wt; 40592-V08H82, RBD-K417N; 40592-V08H59, RBD-N501Y; 40592-V08H82 and RBD-E484K; 40592-V08H84).

    Techniques: Binding Assay, Neutralization, Variant Assay, Plaque Assay, Two Tailed Test, Expressing, Concentration Assay, Purification, SPR Assay, Mutagenesis

    SARS-CoV-2 spike antibody epitope repertoires recognized by hCoV-2IG. SARS-CoV-2 spike GFPDL analyses of IgG antibodies in six batches of hCoV-2IG. (A) Number of IgG bound phage clones selected using SARS-CoV-2 spike GFPDL on six lots of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6). (B) Epitope repertoires of IgG antibody in hCoV-2IG batches and their alignment to the spike protein of SARS-CoV-2. Graphical distribution of representative clones with a frequency of > 2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the alignment of peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value is shown enclosed in a black box beneath the alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and a similar number of phage clones and epitope repertoire was observed in both phage display analysis. (C) SPR binding of hCOV-2IG (n=6; in red), control pre-pandemic 2019-IVIG (n=16; in black) and convalescent plasma (n=9; in blue) with SARS-CoV-2 spike antigenic site peptides identified using GFPDL analysis in Fig. 1B . The amino acid designation is based on the SARS-CoV-2 spike protein sequence ( Fig. S1 ). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution of CP, and 1mg/mL of 2019-IVIG or hCoV-2IG. The numbers above the peptides show the mean value for each respective group antibody binding to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue). All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Journal: bioRxiv

    Article Title: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19

    doi: 10.1101/2021.03.19.436183

    Figure Lengend Snippet: SARS-CoV-2 spike antibody epitope repertoires recognized by hCoV-2IG. SARS-CoV-2 spike GFPDL analyses of IgG antibodies in six batches of hCoV-2IG. (A) Number of IgG bound phage clones selected using SARS-CoV-2 spike GFPDL on six lots of hCoV-2IG (hCoV-2IG-1 to hCoV-2IG-6). (B) Epitope repertoires of IgG antibody in hCoV-2IG batches and their alignment to the spike protein of SARS-CoV-2. Graphical distribution of representative clones with a frequency of > 2, obtained after affinity selection, are shown. The horizontal position and the length of the bars indicate the alignment of peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value is shown enclosed in a black box beneath the alignments. The GFPDL affinity selection data was performed in duplicate (two independent experiments by researcher in the lab, who was blinded to sample identity), and a similar number of phage clones and epitope repertoire was observed in both phage display analysis. (C) SPR binding of hCOV-2IG (n=6; in red), control pre-pandemic 2019-IVIG (n=16; in black) and convalescent plasma (n=9; in blue) with SARS-CoV-2 spike antigenic site peptides identified using GFPDL analysis in Fig. 1B . The amino acid designation is based on the SARS-CoV-2 spike protein sequence ( Fig. S1 ). Total antibody binding is represented in maximum resonance units (RU) in this figure for 10-fold serum dilution of CP, and 1mg/mL of 2019-IVIG or hCoV-2IG. The numbers above the peptides show the mean value for each respective group antibody binding to the peptide and is color-coded (6 hCOV-2IG in red, 16 2019-IVIG in black, and 9 CPs in blue). All SPR experiments were performed twice and the researchers performing the assay were blinded to sample identity. The variations for duplicate runs of SPR was

    Article Snippet: Proteins Recombinant SARS-CoV-2 spike receptor binding domain (RBD) and its mutants were purchased from Sino Biologicals (RBD-wt; 40592-V08H82, RBD-K417N; 40592-V08H59, RBD-N501Y; 40592-V08H82 and RBD-E484K; 40592-V08H84).

    Techniques: Clone Assay, Selection, Sequencing, Isolation, SPR Assay, Binding Assay