sars cov2 s1  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike S1 His Recombinant Protein HPLC verified COVID 19 Spike S1 Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV spike protein S1 Subunit YP 009724390 1 Val16 Arg685 was expressed with a polyhistidine tag at the C terminus
    Catalog Number:
    40591-V08H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
    Buy from Supplier


    Structured Review

    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV spike protein S1 Subunit YP 009724390 1 Val16 Arg685 was expressed with a polyhistidine tag at the C terminus
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    Images

    1) Product Images from "Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors"

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

    Journal: Small Methods

    doi: 10.1002/smtd.202001031

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

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

    2) Product Images from "Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates"

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    Journal: Vaccines

    doi: 10.3390/vaccines9040307

    Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) ( a ). BALB/c mice ( n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S ΔTM , pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies ( b ). All data are represented as individual values. ** p
    Figure Legend Snippet: Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) ( a ). BALB/c mice ( n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S ΔTM , pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies ( b ). All data are represented as individual values. ** p

    Techniques Used: Expressing, Mouse Assay, Plasmid Preparation

    3) Product Images from "SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence"

    Article Title: SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiaa479

    Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A , Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B , Laboratory-confirmed viral infections (see Supplementary Table 2 ) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.
    Figure Legend Snippet: Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A , Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B , Laboratory-confirmed viral infections (see Supplementary Table 2 ) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.

    Techniques Used: Infection, Concentration Assay, Binding Assay

    4) Product Images from "Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals"

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    Journal: medRxiv

    doi: 10.1101/2021.02.07.21251311

    Few antigen-specific ASC induced in circulation after the second vaccine dose in SARS-CoV-2-experienced adults. ( A and B ). Antibody-secreting cell (ASC) ELISpots for a SARS-CoV-2-naive ( A ) or SARS-CoV-2-experienced ( B ) adult one week after each dose of vaccine. ( C to F ) Summary statistics for ELISpot assays. For each panel, S1 (left), S2 (middle), or RBD (right) antigens for IgG or IgA are represented, at one week after first dose ( C and D ) or second dose ( E and F ). Nominal P values from Wilcoxon tests. ( G to I ) ELISpot results for SARS-CoV-2-naive (left) or SARS-CoV-2-experienced (right) adults for S1 ( G ), S2 ( H ), or RBD ( I ). Connected lines indicate repeated measurements from the same participants. Nominal P values from paired t-tests. ( J and K ) Kendall correlations for ELISpot results one week after the first vaccination ( J ) or one week after the second vaccination ( K ). Correlations shown for comparisons with nominal P values
    Figure Legend Snippet: Few antigen-specific ASC induced in circulation after the second vaccine dose in SARS-CoV-2-experienced adults. ( A and B ). Antibody-secreting cell (ASC) ELISpots for a SARS-CoV-2-naive ( A ) or SARS-CoV-2-experienced ( B ) adult one week after each dose of vaccine. ( C to F ) Summary statistics for ELISpot assays. For each panel, S1 (left), S2 (middle), or RBD (right) antigens for IgG or IgA are represented, at one week after first dose ( C and D ) or second dose ( E and F ). Nominal P values from Wilcoxon tests. ( G to I ) ELISpot results for SARS-CoV-2-naive (left) or SARS-CoV-2-experienced (right) adults for S1 ( G ), S2 ( H ), or RBD ( I ). Connected lines indicate repeated measurements from the same participants. Nominal P values from paired t-tests. ( J and K ) Kendall correlations for ELISpot results one week after the first vaccination ( J ) or one week after the second vaccination ( K ). Correlations shown for comparisons with nominal P values

    Techniques Used: Enzyme-linked Immunospot

    SARS-CoV-2-experienced individuals’ robust anti-S1 binding and neutralizing antibodies responses after vaccination. ( A ) Anti-S1 IgG serum antibody titers over time measured in days relative to the first vaccination. ( B ) Anti-S1 IgG titer one week after second vaccination for each cohort ( P =0.06; Wilcoxon test). ( C ) Anti-nucleocapsid IgG serum antibody titers. ( D ) Correlation between fold-change in anti-S1 IgG serum antibody titers, assessed as one week after vaccination compared to baseline, compared to the baseline anti-S1 IgG serum antibody titers, for SARS-CoV-2-experienced adults. ( E ) Example of neutralizing antibody assay shown for the same SARS-CoV-2-experienced participant longitudinally. ( F ) Neutralizing antibody titers one week after the second vaccination ( P =0.19; Wilcoxon test).
    Figure Legend Snippet: SARS-CoV-2-experienced individuals’ robust anti-S1 binding and neutralizing antibodies responses after vaccination. ( A ) Anti-S1 IgG serum antibody titers over time measured in days relative to the first vaccination. ( B ) Anti-S1 IgG titer one week after second vaccination for each cohort ( P =0.06; Wilcoxon test). ( C ) Anti-nucleocapsid IgG serum antibody titers. ( D ) Correlation between fold-change in anti-S1 IgG serum antibody titers, assessed as one week after vaccination compared to baseline, compared to the baseline anti-S1 IgG serum antibody titers, for SARS-CoV-2-experienced adults. ( E ) Example of neutralizing antibody assay shown for the same SARS-CoV-2-experienced participant longitudinally. ( F ) Neutralizing antibody titers one week after the second vaccination ( P =0.19; Wilcoxon test).

    Techniques Used: Binding Assay

    Antibody responses differ based on prior history of COVID-19. ( A ) Anti-S1 IgG antibody titers were assessed for SARS-CoV-2 experienced (purple) and SARS-CoV-2-naive (yellow) adults. Connected lines indicate repeated measurements of the same participants. * P
    Figure Legend Snippet: Antibody responses differ based on prior history of COVID-19. ( A ) Anti-S1 IgG antibody titers were assessed for SARS-CoV-2 experienced (purple) and SARS-CoV-2-naive (yellow) adults. Connected lines indicate repeated measurements of the same participants. * P

    Techniques Used:

    CD4 and CD8 T cell responses and gating strategy. ( A ) Study participant timeline relative to first vaccination. ( B ) Gating scheme for T and B cell populations. ( C ) Non-naive CD8 shown in tSNE projection for SARS-CoV-2-naive (upper) or SARS-CoV-2-experienced (lower) participants. Heatmap shows expression of Ki67. Circled area indicates region corresponding to Cluster 12. ( D ) Non-naive CD8 underwent Phenograph clustering. Protein expression for each cluster for SARS-CoV-2-experienced adults shown for samples taken one week after the second vaccination. ( E and F ) Ki67+CD38+ expression in CD8 T cells by cohort over time measured in days, relative to the individual’s first ( E ) or second ( L ) vaccination. ( G ) Example for Ki67+CD38+ CD8 T cell expression of granzyme B in a SARS-CoV-2-experienced individual. ( H ) Summary data for Ki67+CD38+ CD8 T cell expression of granzyme B. P -values from one-way ANOVA with Tukey’s post test. ( I ) Non-naive CD4 T cells from all samples were merged for tSNE projection. Colors indicate Phenograph clustering. ( J and K ) Protein expression for Phenograph clusters for non-naive CD4 T cells shown for samples at one week following second vaccination in SARS-CoV-2-naive ( J ) or SARS-CoV-2-experienced ( K ) participants. ( L and M ) CD4 T cells shown for expression of Ki67 and CD38 after vaccination over time measured in days, relative to the individual’s first ( L ) or second ( M ) vaccinations. ( N ) Correlation between Ki67+CD38+ CD4 T cells and age one after either first vaccination (left) or second vaccination (right).
    Figure Legend Snippet: CD4 and CD8 T cell responses and gating strategy. ( A ) Study participant timeline relative to first vaccination. ( B ) Gating scheme for T and B cell populations. ( C ) Non-naive CD8 shown in tSNE projection for SARS-CoV-2-naive (upper) or SARS-CoV-2-experienced (lower) participants. Heatmap shows expression of Ki67. Circled area indicates region corresponding to Cluster 12. ( D ) Non-naive CD8 underwent Phenograph clustering. Protein expression for each cluster for SARS-CoV-2-experienced adults shown for samples taken one week after the second vaccination. ( E and F ) Ki67+CD38+ expression in CD8 T cells by cohort over time measured in days, relative to the individual’s first ( E ) or second ( L ) vaccination. ( G ) Example for Ki67+CD38+ CD8 T cell expression of granzyme B in a SARS-CoV-2-experienced individual. ( H ) Summary data for Ki67+CD38+ CD8 T cell expression of granzyme B. P -values from one-way ANOVA with Tukey’s post test. ( I ) Non-naive CD4 T cells from all samples were merged for tSNE projection. Colors indicate Phenograph clustering. ( J and K ) Protein expression for Phenograph clusters for non-naive CD4 T cells shown for samples at one week following second vaccination in SARS-CoV-2-naive ( J ) or SARS-CoV-2-experienced ( K ) participants. ( L and M ) CD4 T cells shown for expression of Ki67 and CD38 after vaccination over time measured in days, relative to the individual’s first ( L ) or second ( M ) vaccinations. ( N ) Correlation between Ki67+CD38+ CD4 T cells and age one after either first vaccination (left) or second vaccination (right).

    Techniques Used: Expressing

    mRNA vaccination induces CD4 and CD8 T cell responses. ( A ) Study schematic. ( B ) Non-naive CD8 T cells from all participants were colored using Phenograph clusters and projected using tSNE. Circled region indicates cluster 12. ( C ) Phenograph cluster abundance for non-naive CD8 T cells was compared using edgeR for all participants before and after the second vaccination. ( D ) Heatmap for non-naive CD8 T cell cluster protein expression for SARS-CoV-2-naive participants after the second vaccination. ( E ) Example of CD8 T cell expression of Ki67 and CD38. ( F ) Summary data for Ki67+CD38+ expression in CD8 T cells by cohort. * P
    Figure Legend Snippet: mRNA vaccination induces CD4 and CD8 T cell responses. ( A ) Study schematic. ( B ) Non-naive CD8 T cells from all participants were colored using Phenograph clusters and projected using tSNE. Circled region indicates cluster 12. ( C ) Phenograph cluster abundance for non-naive CD8 T cells was compared using edgeR for all participants before and after the second vaccination. ( D ) Heatmap for non-naive CD8 T cell cluster protein expression for SARS-CoV-2-naive participants after the second vaccination. ( E ) Example of CD8 T cell expression of Ki67 and CD38. ( F ) Summary data for Ki67+CD38+ expression in CD8 T cells by cohort. * P

    Techniques Used: Expressing

    Poor IgG and IgA ASC responses to second dose in SARS-CoV-2 experienced participants. ( A and B ) Antibody-secreting cell (ASC) ELISpots shown for IgG, IgA, and IgM-producing cells reacting to RBD, S1, or S2 antigens, or total secreted antibody controls.. ( C ) IgM-producing ASC in circulation quantified one week after first vaccination (top) or one week after second vaccination (bottom). Nominal P values from Wilcoxon tests. ( D ) ASC frequencies shown over time measured in days relative to the first vaccination (top) or second vaccination (bottom) for S1, S2, or RBD antigens. ( E and F ) Correlation shown for both cohorts for S1-reactive IgG ASC ( E ) or S2-reactive IgG ASC ( F ) compared to RBD-reactive IgG ASC one week after second vaccination. ( G and H ) Kendall correlation shown for SARS-CoV-2-specific frequencies for SARS-CoV-2-naive or SARS-CoV-2-experienced adults one week after first ( G ) or one week after second ( H ) vaccination. Heatmap colored by Kendall’s tau statistic. Boxes with symbols indicate nominal P value > 0.05.
    Figure Legend Snippet: Poor IgG and IgA ASC responses to second dose in SARS-CoV-2 experienced participants. ( A and B ) Antibody-secreting cell (ASC) ELISpots shown for IgG, IgA, and IgM-producing cells reacting to RBD, S1, or S2 antigens, or total secreted antibody controls.. ( C ) IgM-producing ASC in circulation quantified one week after first vaccination (top) or one week after second vaccination (bottom). Nominal P values from Wilcoxon tests. ( D ) ASC frequencies shown over time measured in days relative to the first vaccination (top) or second vaccination (bottom) for S1, S2, or RBD antigens. ( E and F ) Correlation shown for both cohorts for S1-reactive IgG ASC ( E ) or S2-reactive IgG ASC ( F ) compared to RBD-reactive IgG ASC one week after second vaccination. ( G and H ) Kendall correlation shown for SARS-CoV-2-specific frequencies for SARS-CoV-2-naive or SARS-CoV-2-experienced adults one week after first ( G ) or one week after second ( H ) vaccination. Heatmap colored by Kendall’s tau statistic. Boxes with symbols indicate nominal P value > 0.05.

    Techniques Used:

    5) Product Images from "A New Saliva-Based Lateral-Flow SARS-CoV-2 IgG Antibody Test for mRNA Vaccination"

    Article Title: A New Saliva-Based Lateral-Flow SARS-CoV-2 IgG Antibody Test for mRNA Vaccination

    Journal: medRxiv

    doi: 10.1101/2021.06.11.21258769

    ROC curves for the nLF SARS-CoV-2 IgG assay. a , ROC curve for the nLF SARS-CoV-2 anti-S1 IgG assay. The ROC curve is based on 34 unvaccinated healthy saliva samples without prior COVID-19 infection, 20 saliva samples from PCR confirmed COVID-19 patients (16 samples were collected ≥ 15 days post symptom onset, 1 sample was collected 11 days post symptom onset, and 3 samples had no information regarding the number of days between symptom onset to sample collection), and 205 vaccinated (with mRNA vaccines from Pfizer/BioNTech and Moderna) saliva samples (collected ≥ 19 days post 1st vaccine shot from subjects without prior COVID-19 infection). A cut-off value of 0.921 T-line intensity unit was determined via the ROC curve under the criteria of 100% specificity and 99.6% sensitivity. b , ROC curve for the nLF SARS-CoV-2 anti-NCP IgG assay. The ROC curve is based on 12 negative and 20 PCR-positive COVID-19 saliva samples. The ROC analysis gave a cutoff value of 1.004 intensity unit under the criteria of 100% specificity and 100% sensitivity.
    Figure Legend Snippet: ROC curves for the nLF SARS-CoV-2 IgG assay. a , ROC curve for the nLF SARS-CoV-2 anti-S1 IgG assay. The ROC curve is based on 34 unvaccinated healthy saliva samples without prior COVID-19 infection, 20 saliva samples from PCR confirmed COVID-19 patients (16 samples were collected ≥ 15 days post symptom onset, 1 sample was collected 11 days post symptom onset, and 3 samples had no information regarding the number of days between symptom onset to sample collection), and 205 vaccinated (with mRNA vaccines from Pfizer/BioNTech and Moderna) saliva samples (collected ≥ 19 days post 1st vaccine shot from subjects without prior COVID-19 infection). A cut-off value of 0.921 T-line intensity unit was determined via the ROC curve under the criteria of 100% specificity and 99.6% sensitivity. b , ROC curve for the nLF SARS-CoV-2 anti-NCP IgG assay. The ROC curve is based on 12 negative and 20 PCR-positive COVID-19 saliva samples. The ROC analysis gave a cutoff value of 1.004 intensity unit under the criteria of 100% specificity and 100% sensitivity.

    Techniques Used: Infection, Polymerase Chain Reaction

    Detection limit of anti-spike IgG in serum by nLF and MidaSpot. Purified recombinant anti-SARS-CoV-2 spike protein IgG was spiked into a negative serum sample and conducted serial dilutions (4X to 1024X dilutions) to determine the detection limit by nLF and MidaSpot. a , Detection limit of anti-spike IgG in serum by MidaSpot. Nirmidas’ MidaSpot™ COVID-19 rapid Antibody Combo Detection Kit was approved by FDA EUA for POC testing. Based on the testing instruction, 10 µl serum sample was applied to the sample window followed by adding 4 drops (∼ 100 µl) of running buffer, and after 20 min the result was recorded. MidaSpot presents positive signals for the 4X and lower dilutions. b , Detection limit of anti-spike IgG in serum by nLF. The nLF (see Method) is able to detect anti-spike IgG even in the 256x diluted serum sample, affording a ∼ 64 times lower detection limit than the conventional LF approach owing to the removal of excess, non-specific serum IgG by nLF.
    Figure Legend Snippet: Detection limit of anti-spike IgG in serum by nLF and MidaSpot. Purified recombinant anti-SARS-CoV-2 spike protein IgG was spiked into a negative serum sample and conducted serial dilutions (4X to 1024X dilutions) to determine the detection limit by nLF and MidaSpot. a , Detection limit of anti-spike IgG in serum by MidaSpot. Nirmidas’ MidaSpot™ COVID-19 rapid Antibody Combo Detection Kit was approved by FDA EUA for POC testing. Based on the testing instruction, 10 µl serum sample was applied to the sample window followed by adding 4 drops (∼ 100 µl) of running buffer, and after 20 min the result was recorded. MidaSpot presents positive signals for the 4X and lower dilutions. b , Detection limit of anti-spike IgG in serum by nLF. The nLF (see Method) is able to detect anti-spike IgG even in the 256x diluted serum sample, affording a ∼ 64 times lower detection limit than the conventional LF approach owing to the removal of excess, non-specific serum IgG by nLF.

    Techniques Used: Purification, Recombinant

    6) Product Images from "Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2"

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

    Journal: ACS Infectious Diseases

    doi: 10.1021/acsinfecdis.1c00070

    Concentration-dependent inhibition of SARS-CoV-S1S2 binding to ACE2 by representative compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-S1S2 (His-tagged, 1 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay by selected representative dye and nondye compounds. Data and fit as before ( Figure 3 ). Most compounds including several DRI-C compounds show similar activity against SARS-CoV (i.e., SARS-CoV-1) as against SARS-CoV-2 raising the possibility of broad-spectrum activity.
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-S1S2 binding to ACE2 by representative compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-S1S2 (His-tagged, 1 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay by selected representative dye and nondye compounds. Data and fit as before ( Figure 3 ). Most compounds including several DRI-C compounds show similar activity against SARS-CoV (i.e., SARS-CoV-1) as against SARS-CoV-2 raising the possibility of broad-spectrum activity.

    Techniques Used: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Concentration–response curves for binding of CoV spike protein domains to human ACE2 in cell-free ELISA-type assays. Binding curves and corresponding EC 50 ’s are shown for SARS-CoV-2 (RBD and S1), SARS-CoV (S1 S2), and HCoV-NL63 (S1). They were obtained using Fc-conjugated hACE2 coated on the plate and His-tagged S1, S1S2, or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).
    Figure Legend Snippet: Concentration–response curves for binding of CoV spike protein domains to human ACE2 in cell-free ELISA-type assays. Binding curves and corresponding EC 50 ’s are shown for SARS-CoV-2 (RBD and S1), SARS-CoV (S1 S2), and HCoV-NL63 (S1). They were obtained using Fc-conjugated hACE2 coated on the plate and His-tagged S1, S1S2, or RBD added in increasing amounts as shown with the amount bound detected using an anti-His–HRP conjugate (mean ± SD for two experiments in duplicates).

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

    Concentration-dependent inhibition of SARS-CoV-2 pseudovirus entry (BacMam) into hACE2 expressing host cells by selected compounds. Quantification of entry of pseudoviruses bearing the SARS-CoV-2 S protein (plus green fluorescent protein reporters; BacMam-based) in ACE2 (plus red fluorescence)-expressing host cells (HEK293T). Representative images (bottom row) and their quantification for pseudovirus (green) and ACE2 expression (red) using ImageJ (top row) are shown from one experiment for CgRd and DRI-C23041 in (A) and (B), respectively; average data from three experiments fitted with typical concentration–response curves are shown in (C). The amount of green present is proportional with the number of infected cells as green fluorescence is expressed only in pseudovirus infected cells, while amount of red is proportional with the number of ACE2-expressing cells. The organic dye CgRd (A), but especially DRI-C23041 (B) showed concentration-dependent inhibition with activities corresponding to low micromolar IC 50 values, whereas hydroxychloroquine (HCQ) showed no effect (C).
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2 pseudovirus entry (BacMam) into hACE2 expressing host cells by selected compounds. Quantification of entry of pseudoviruses bearing the SARS-CoV-2 S protein (plus green fluorescent protein reporters; BacMam-based) in ACE2 (plus red fluorescence)-expressing host cells (HEK293T). Representative images (bottom row) and their quantification for pseudovirus (green) and ACE2 expression (red) using ImageJ (top row) are shown from one experiment for CgRd and DRI-C23041 in (A) and (B), respectively; average data from three experiments fitted with typical concentration–response curves are shown in (C). The amount of green present is proportional with the number of infected cells as green fluorescence is expressed only in pseudovirus infected cells, while amount of red is proportional with the number of ACE2-expressing cells. The organic dye CgRd (A), but especially DRI-C23041 (B) showed concentration-dependent inhibition with activities corresponding to low micromolar IC 50 values, whereas hydroxychloroquine (HCQ) showed no effect (C).

    Techniques Used: Concentration Assay, Inhibition, Expressing, Fluorescence, Infection

    Concentration-dependent inhibition of SARS-CoV-2 pseudovirus (VSV-Δ G ) entry into hACE2/Furin expressing host cells by selected compounds. Entry of VSV-ΔG pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero-E6) was quantified via GFP fluorescence in a live imaging system (Incucyte). CgRd and DRI-C23041 showed concentration-dependent inhibition with IC 50 values consistent with the previous assay ( Figure 7 ), whereas the negative control sunset yellow (SY FD C #6) showed no significant effect.
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2 pseudovirus (VSV-Δ G ) entry into hACE2/Furin expressing host cells by selected compounds. Entry of VSV-ΔG pseudoviruses bearing the SARS-CoV-2 S protein (plus GFP reporters) in ACE2/Furin overexpressing host cells (Vero-E6) was quantified via GFP fluorescence in a live imaging system (Incucyte). CgRd and DRI-C23041 showed concentration-dependent inhibition with IC 50 values consistent with the previous assay ( Figure 7 ), whereas the negative control sunset yellow (SY FD C #6) showed no significant effect.

    Techniques Used: Concentration Assay, Inhibition, Expressing, Fluorescence, Imaging, Negative Control

    Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-2-RBD (His-tagged, 0.5 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay with dye (A) and nondye (B) compounds tested. The promiscuous PPI inhibitor erythrosine B (ErB) and the food colorant FD C yellow no. 6 (sunset yellow, SY) were included as a positive and negative controls, respectively. Data are mean ± SD from two experiments in duplicates and were fitted with standard sigmoid curves for IC 50 determination. Estimated IC 50 ’s are shown in the legend indicating that while suramin and chloroquine were completely inactive (IC 50 > 500 μM), several of our in-house compounds including organic dyes (CgRd, DV1, and others) as well as proprietary DRI-C compounds (e.g., DRI-C23041, DRI-C91005) showed promising activity, some even at submicromolar levels (IC 50
    Figure Legend Snippet: Concentration-dependent inhibition of SARS-CoV-2-S-RBD binding to ACE2 by compounds of the present study. Concentration–response curves obtained for the inhibition of the PPI between SARS-CoV-2-RBD (His-tagged, 0.5 μg/mL) and hACE2 (Fc-conjugated, 1 μg/mL) in cell-free ELISA-type assay with dye (A) and nondye (B) compounds tested. The promiscuous PPI inhibitor erythrosine B (ErB) and the food colorant FD C yellow no. 6 (sunset yellow, SY) were included as a positive and negative controls, respectively. Data are mean ± SD from two experiments in duplicates and were fitted with standard sigmoid curves for IC 50 determination. Estimated IC 50 ’s are shown in the legend indicating that while suramin and chloroquine were completely inactive (IC 50 > 500 μM), several of our in-house compounds including organic dyes (CgRd, DV1, and others) as well as proprietary DRI-C compounds (e.g., DRI-C23041, DRI-C91005) showed promising activity, some even at submicromolar levels (IC 50

    Techniques Used: Concentration Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    Identification of the binding partner by protein thermal shift. Differential scanning fluorimetry assay indicating SARS-CoV-2 RBD and not ACE2 as the binding partner of the present SMI compounds. The presence of Congo red (top) or DRI-C23041 (bottom) at 10 μM caused clear shifts in the melting temperature of the protein for RBD as indicated by the derivatives d F /d T (left; purple vs blue line), but not for hACE2 (right) (smaller insets are normalized fluorescence F data).
    Figure Legend Snippet: Identification of the binding partner by protein thermal shift. Differential scanning fluorimetry assay indicating SARS-CoV-2 RBD and not ACE2 as the binding partner of the present SMI compounds. The presence of Congo red (top) or DRI-C23041 (bottom) at 10 μM caused clear shifts in the melting temperature of the protein for RBD as indicated by the derivatives d F /d T (left; purple vs blue line), but not for hACE2 (right) (smaller insets are normalized fluorescence F data).

    Techniques Used: Binding Assay, Fluorimetry Assay, Fluorescence

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.07.24.220715

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

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

    8) Product Images from "Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor"

    Article Title: Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c02823

    Detection of SARS-CoV-2 antigen protein. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 spike protein. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in PBS and (C) related dose-dependent response curve ( V DS = 0.01 V). Graphene-based FET without SARS-CoV-2 antibody is presented as negative control. (D) Selective response of COVID-19 FET sensor toward target SARS-CoV-2 antigen protein and MERS-CoV protein. (E) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in UTM and (F) related dose-dependent response curve.
    Figure Legend Snippet: Detection of SARS-CoV-2 antigen protein. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 spike protein. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in PBS and (C) related dose-dependent response curve ( V DS = 0.01 V). Graphene-based FET without SARS-CoV-2 antibody is presented as negative control. (D) Selective response of COVID-19 FET sensor toward target SARS-CoV-2 antigen protein and MERS-CoV protein. (E) Real-time response of COVID-19 FET toward SARS-CoV-2 antigen protein in UTM and (F) related dose-dependent response curve.

    Techniques Used: Negative Control

    Detection of cultured SARS-CoV-2 virus. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 cultured virus. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 cultured virus and (C) related dose-dependent response curve.
    Figure Legend Snippet: Detection of cultured SARS-CoV-2 virus. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 cultured virus. (B) Real-time response of COVID-19 FET toward SARS-CoV-2 cultured virus and (C) related dose-dependent response curve.

    Techniques Used: Cell Culture

    Electrical characterization of pristine, PBASE-modified, and SARS-CoV-2 spike antibody-immobilized graphene. (A) Schematic diagram of the aqueous-solution-gated FET (COVID-19 FET sensor) configuration using the antibody-conjugated graphene. (B) I DS – V DS output curves of the antibody-conjugated FET with various gating voltages from 0 to −1.5 V in steps of −0.3 V. I DS negatively increased as V GS negatively increased. (C) Current–voltage ( I–V ) characteristics of the graphene-based device of each functionalization process for the antibody modification. (D) Measurement of transfer curves of the COVID-19 FET sensor in steps of the antibody conjugation ( V DS = 0.01 V).
    Figure Legend Snippet: Electrical characterization of pristine, PBASE-modified, and SARS-CoV-2 spike antibody-immobilized graphene. (A) Schematic diagram of the aqueous-solution-gated FET (COVID-19 FET sensor) configuration using the antibody-conjugated graphene. (B) I DS – V DS output curves of the antibody-conjugated FET with various gating voltages from 0 to −1.5 V in steps of −0.3 V. I DS negatively increased as V GS negatively increased. (C) Current–voltage ( I–V ) characteristics of the graphene-based device of each functionalization process for the antibody modification. (D) Measurement of transfer curves of the COVID-19 FET sensor in steps of the antibody conjugation ( V DS = 0.01 V).

    Techniques Used: Modification, Conjugation Assay

    Detection of SARS-CoV-2 virus from clinical samples. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 virus from COVID-19 patients. (B,C) Comparison of response signal between normal samples and patient ones. (D) Real-time response of COVID-19 FET toward SARS-CoV-2 clinical sample and (C) related dose-dependent response curve.
    Figure Legend Snippet: Detection of SARS-CoV-2 virus from clinical samples. (A) Schematic diagram for the COVID-19 FET sensor for detection of SARS-CoV-2 virus from COVID-19 patients. (B,C) Comparison of response signal between normal samples and patient ones. (D) Real-time response of COVID-19 FET toward SARS-CoV-2 clinical sample and (C) related dose-dependent response curve.

    Techniques Used:

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

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.599587

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

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

    10) Product Images from "Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in Rhesus macaques"

    Article Title: Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in Rhesus macaques

    Journal: bioRxiv

    doi: 10.1101/2020.03.13.990036

    Graphic outline of experimental design and sample collection. Five male rhesus macaques ( Macaca Mulatta ) between the ages of 3 and 5 years were inoculated with 10 6 TCID 50 /ml SARS-CoV-2. Two rhesus macaques via ocular conjunctival route named CJ-1 and CJ-2, one was inoculated via intratracheal route named IT-1, two were inoculated via intragastric route in sequence named IG-1 and IG-2, respectively. The macaques were observed daily for clinical signs (body weight temperature were tested as shown). On 0, 1, 3, 5, and 7 dpi, the conjunctival, nasal, throat and anal swabs were collected. CJ-1, IT-1, and IG-1 were euthanized and necropsied on 7 dpi. Tissues were collected to analysis the virus distributions. All sera were collected on 0, 7, 14 and 21 dpi for serologic detection to exam the SARS-CoV-2 specific IgG antibodies.
    Figure Legend Snippet: Graphic outline of experimental design and sample collection. Five male rhesus macaques ( Macaca Mulatta ) between the ages of 3 and 5 years were inoculated with 10 6 TCID 50 /ml SARS-CoV-2. Two rhesus macaques via ocular conjunctival route named CJ-1 and CJ-2, one was inoculated via intratracheal route named IT-1, two were inoculated via intragastric route in sequence named IG-1 and IG-2, respectively. The macaques were observed daily for clinical signs (body weight temperature were tested as shown). On 0, 1, 3, 5, and 7 dpi, the conjunctival, nasal, throat and anal swabs were collected. CJ-1, IT-1, and IG-1 were euthanized and necropsied on 7 dpi. Tissues were collected to analysis the virus distributions. All sera were collected on 0, 7, 14 and 21 dpi for serologic detection to exam the SARS-CoV-2 specific IgG antibodies.

    Techniques Used: Sequencing

    Clinical features, viral distributions and antibody detection in the sera from the rhesus macaques inoculated with SARS-CoV-2 via three routes. Clinical signs including body weight (A) and temperature (B) were observed. The viral load of the conjunctival, nasal, throat, and anal swabs specimens (C) from the five inoculated macaques on 0, 1, 3, 5, and 7 dpi. The comparison of viral distributionsin the majority of organs and tissues (D) from CJ-1 and IT-1 on 7 dpi. The darker the blue color, the higher the viral load. The specific IgG antibody against SARS-CoV-2 in the sera of the inoculated-macaques were tested by ELISA on 0, 7, 14, and 21 dpi (E). According to unpaired Welch’s t -test, the specific IgG antibody in the sera of conjunctival inoculated macaque exhibited a significant increase compared with prior to inoculation (**p=0.0027) and 21 dpi (**p=0.0039) . CJ-1 and CJ-2 were the two macaques that inoculated via conjunctival route, IT-1 was the macaque that inoculated via intratracheal route. IG-1 and IG-2 were the two macaques that inoculated via intragastric route. ND, not detected. NC, negative control (unpaired Welch’s t -test, ** p
    Figure Legend Snippet: Clinical features, viral distributions and antibody detection in the sera from the rhesus macaques inoculated with SARS-CoV-2 via three routes. Clinical signs including body weight (A) and temperature (B) were observed. The viral load of the conjunctival, nasal, throat, and anal swabs specimens (C) from the five inoculated macaques on 0, 1, 3, 5, and 7 dpi. The comparison of viral distributionsin the majority of organs and tissues (D) from CJ-1 and IT-1 on 7 dpi. The darker the blue color, the higher the viral load. The specific IgG antibody against SARS-CoV-2 in the sera of the inoculated-macaques were tested by ELISA on 0, 7, 14, and 21 dpi (E). According to unpaired Welch’s t -test, the specific IgG antibody in the sera of conjunctival inoculated macaque exhibited a significant increase compared with prior to inoculation (**p=0.0027) and 21 dpi (**p=0.0039) . CJ-1 and CJ-2 were the two macaques that inoculated via conjunctival route, IT-1 was the macaque that inoculated via intratracheal route. IG-1 and IG-2 were the two macaques that inoculated via intragastric route. ND, not detected. NC, negative control (unpaired Welch’s t -test, ** p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control

    Compare the lesions in lungs from CJ-1 and IT-1 by radiographic alterations, histopathological and Immunohistochemical observation. The anterior-posterior and right lateral chest radiographs (A) from rhesus macaque imaged prior to SARS-CoV-2 inoculation (day 0) and 7 dpi. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle); obscures costophrenic angle (red arrows); patchy lesions (blue circle). Positional indicators are included (R=right). The histopathological and immunohistochemical observations in the lungs (B). Both the two macaques exhibited interstitial pneumonia with thickened alveolar septa, filtration of inflammatory cells mainly including lymphocytes and macrophages, some amounts of exudation (red arrows) in the alveolar cavities on 7dpi. Conjunctival route caused relatively mild pneumonia. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed primarily in the alveolar epithelia (black arrows) and the detached-degenerative cellular debris (green arrows). The H E stained-sections under 400 magnification were the fractionated gain (black frame) of these sections under 100 magnification. The IHC section showed the same field with the black frame section under 400 magnification. Black scale bar = 100 μm, red scale bar = 50 μm.
    Figure Legend Snippet: Compare the lesions in lungs from CJ-1 and IT-1 by radiographic alterations, histopathological and Immunohistochemical observation. The anterior-posterior and right lateral chest radiographs (A) from rhesus macaque imaged prior to SARS-CoV-2 inoculation (day 0) and 7 dpi. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle); obscures costophrenic angle (red arrows); patchy lesions (blue circle). Positional indicators are included (R=right). The histopathological and immunohistochemical observations in the lungs (B). Both the two macaques exhibited interstitial pneumonia with thickened alveolar septa, filtration of inflammatory cells mainly including lymphocytes and macrophages, some amounts of exudation (red arrows) in the alveolar cavities on 7dpi. Conjunctival route caused relatively mild pneumonia. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed primarily in the alveolar epithelia (black arrows) and the detached-degenerative cellular debris (green arrows). The H E stained-sections under 400 magnification were the fractionated gain (black frame) of these sections under 100 magnification. The IHC section showed the same field with the black frame section under 400 magnification. Black scale bar = 100 μm, red scale bar = 50 μm.

    Techniques Used: Immunohistochemistry, Filtration, Staining

    11) Product Images from "Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies"

    Article Title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422550

    The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and
    Figure Legend Snippet: The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and

    Techniques Used:

    Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .
    Figure Legend Snippet: Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .

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

    The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.
    Figure Legend Snippet: The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.

    Techniques Used: Sequencing, Mutagenesis

    12) Product Images from "Age‐related rhesus macaque models of COVID‐19, et al. Age‐related rhesus macaque models of COVID‐19"

    Article Title: Age‐related rhesus macaque models of COVID‐19, et al. Age‐related rhesus macaque models of COVID‐19

    Journal: Animal Models and Experimental Medicine

    doi: 10.1002/ame2.12108

    Viral load of the SARS‐CoV‐2‐infected rhesus macaque model. A. Average viral loads of swabs from the younger group (YG, n = 3, red line) monkeys. B. Average viral load of swabs from the elder group (EG, n = 2, blue line) monkeys. Viral loads of nasal, throat, and anal swab specimens collected from the inoculated macaques on 0, 3, 5, 7, 9, 11, and 14 dpi. C. Viral loads in varies lobe of lung tissue from YG and EG monkeys at day 7 post‐inoculation. RNA was extracted and viral load was determined by qRT‐PCR. All data are presented as mean ± SEM
    Figure Legend Snippet: Viral load of the SARS‐CoV‐2‐infected rhesus macaque model. A. Average viral loads of swabs from the younger group (YG, n = 3, red line) monkeys. B. Average viral load of swabs from the elder group (EG, n = 2, blue line) monkeys. Viral loads of nasal, throat, and anal swab specimens collected from the inoculated macaques on 0, 3, 5, 7, 9, 11, and 14 dpi. C. Viral loads in varies lobe of lung tissue from YG and EG monkeys at day 7 post‐inoculation. RNA was extracted and viral load was determined by qRT‐PCR. All data are presented as mean ± SEM

    Techniques Used: Infection, Quantitative RT-PCR

    Hematological analysis in rhesus macaques inoculated with SARS‐CoV‐2. A. The counts of white blood cells (WBC) were analysed. B. The percentage and counts of monocytes were determined. C. The percentage and counts of lymphocytes were detected. D. The percentage and counts of CD3 + CD8 + T cells, CD3 + CD4 + T cells were shown. YG (red line) and EG (blue line) were indicated in the upper right corner of each panel. All data are presented as mean ± SEM
    Figure Legend Snippet: Hematological analysis in rhesus macaques inoculated with SARS‐CoV‐2. A. The counts of white blood cells (WBC) were analysed. B. The percentage and counts of monocytes were determined. C. The percentage and counts of lymphocytes were detected. D. The percentage and counts of CD3 + CD8 + T cells, CD3 + CD4 + T cells were shown. YG (red line) and EG (blue line) were indicated in the upper right corner of each panel. All data are presented as mean ± SEM

    Techniques Used:

    The comparison of lesions in the lung between younger group (YG) and elder group (EG) by radiographic alterations, histopathological and immunohistochemical (IHC) observation of the SARS‐CoV‐2‐inoculated‐rhesus macaque. A. Anterior‐posterior thoracic X‐rays from of rhesus macaque imaged prior to SARS‐CoV‐2 inoculation (day 0) and on 7 dpi of YG and 5 dpi of EG. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle). Positional indicators are included (R = right). B. Histopathological changes in lungs from YG and EG. Lung tissue was collected and stained with hematoxylin and eosin. Black scale bar = 40 µm. IHC staining demonstrated that SARS‐CoV‐2 antigens were mainly in the epithelial cells and macrophages. SARS‐CoV‐2 antigens were indicated by red arrows. Red scale bar = 50 µm
    Figure Legend Snippet: The comparison of lesions in the lung between younger group (YG) and elder group (EG) by radiographic alterations, histopathological and immunohistochemical (IHC) observation of the SARS‐CoV‐2‐inoculated‐rhesus macaque. A. Anterior‐posterior thoracic X‐rays from of rhesus macaque imaged prior to SARS‐CoV‐2 inoculation (day 0) and on 7 dpi of YG and 5 dpi of EG. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle). Positional indicators are included (R = right). B. Histopathological changes in lungs from YG and EG. Lung tissue was collected and stained with hematoxylin and eosin. Black scale bar = 40 µm. IHC staining demonstrated that SARS‐CoV‐2 antigens were mainly in the epithelial cells and macrophages. SARS‐CoV‐2 antigens were indicated by red arrows. Red scale bar = 50 µm

    Techniques Used: Immunohistochemistry, Staining

    13) Product Images from "Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants with a novel competitive multiplex assay"

    Article Title: Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants with a novel competitive multiplex assay

    Journal: medRxiv

    doi: 10.1101/2021.03.20.21254037

    Principle and validation of the SARS-CoV-2 inhibition assay A . Schematic representation of the RBD multiplex assay: Anti-SARS-CoV-2 RBD antibodies compete with biotin-conjugated ACE2 for binding to RBD variants coupled to magnetic microspheres. B . Inhibition of the RBD-ACE2 interaction by COVID-19 subjects recovered from SARS-CoV-2 infection (COVID-19 A, B and C) vs Mean ±S.D of ( n =7) COVID-19 negative samples, with a decrease in MFI observed as anti-SARS-CoV-2 neutralizing antibodies present in the sample block the ACE2 binding RBD coupled to magnetic microspheres. C . Inhibition of the SARS-CoV-2 RBD–ACE2 interaction by plasma from convalescent COVID-19 subjects, with the determined cut-off at 20% inhibition indicated by the dotted line. Illness severity was severe for example COVID-19 subject A (orange-squares) moderate for B (yellow-triangles) and mild for subject C (green-inverted triangles). Illustrations were created using BioRender. D . Plasma samples from subjects recovered from SARS-CoV-2 infection ( n = 37) and SARS-CoV-2 negative healthy controls ( n = 7) were tested for inhibition of the RBD-ACE2 interaction and E . the Spike (S1)-ACE2 interaction. SARS-CoV-2 positive subjects are colored based on COVID-19 illness severity. Orange Squares=Severe, Yellow Triangles=Moderate, Green Inverted Triangles=Mild. Grey = SARS-CoV-2 negative controls. F . Spearman’ s Correlation between relative Spike (S1) and RBD IC 50 . percentage inhibition values. G . Spearman’ s Correlation of the percentage ACE2 inhibition at a 1 in 100 dilution of plasma to RBD and the relative IC 50 . value. H . Spearman’ s Correlation of Multiplex ACE-RBD inhibition assay IC 50 values and the titer obtained with the virus microneutralization assay. I . Spearman’ s Correlation of Multiplex ACE-Spike (S1) inhibition assay IC 50 .values and the titer obtained with the virus microneutralization assay. J . Spearman’ s Correlation of ACE-RBD inhibition and RBD Binding K . RBD binding of control mAbs L . Percentage ACE2 inhibition of control mAbs.
    Figure Legend Snippet: Principle and validation of the SARS-CoV-2 inhibition assay A . Schematic representation of the RBD multiplex assay: Anti-SARS-CoV-2 RBD antibodies compete with biotin-conjugated ACE2 for binding to RBD variants coupled to magnetic microspheres. B . Inhibition of the RBD-ACE2 interaction by COVID-19 subjects recovered from SARS-CoV-2 infection (COVID-19 A, B and C) vs Mean ±S.D of ( n =7) COVID-19 negative samples, with a decrease in MFI observed as anti-SARS-CoV-2 neutralizing antibodies present in the sample block the ACE2 binding RBD coupled to magnetic microspheres. C . Inhibition of the SARS-CoV-2 RBD–ACE2 interaction by plasma from convalescent COVID-19 subjects, with the determined cut-off at 20% inhibition indicated by the dotted line. Illness severity was severe for example COVID-19 subject A (orange-squares) moderate for B (yellow-triangles) and mild for subject C (green-inverted triangles). Illustrations were created using BioRender. D . Plasma samples from subjects recovered from SARS-CoV-2 infection ( n = 37) and SARS-CoV-2 negative healthy controls ( n = 7) were tested for inhibition of the RBD-ACE2 interaction and E . the Spike (S1)-ACE2 interaction. SARS-CoV-2 positive subjects are colored based on COVID-19 illness severity. Orange Squares=Severe, Yellow Triangles=Moderate, Green Inverted Triangles=Mild. Grey = SARS-CoV-2 negative controls. F . Spearman’ s Correlation between relative Spike (S1) and RBD IC 50 . percentage inhibition values. G . Spearman’ s Correlation of the percentage ACE2 inhibition at a 1 in 100 dilution of plasma to RBD and the relative IC 50 . value. H . Spearman’ s Correlation of Multiplex ACE-RBD inhibition assay IC 50 values and the titer obtained with the virus microneutralization assay. I . Spearman’ s Correlation of Multiplex ACE-Spike (S1) inhibition assay IC 50 .values and the titer obtained with the virus microneutralization assay. J . Spearman’ s Correlation of ACE-RBD inhibition and RBD Binding K . RBD binding of control mAbs L . Percentage ACE2 inhibition of control mAbs.

    Techniques Used: Inhibition, Multiplex Assay, Binding Assay, Infection, Blocking Assay, Microneutralization Assay

    14) Product Images from "Longitudinal antibody repertoire in “mild” versus “severe” COVID-19 patients reveals immune markers associated with disease severity and resolution"

    Article Title: Longitudinal antibody repertoire in “mild” versus “severe” COVID-19 patients reveals immune markers associated with disease severity and resolution

    Journal: Science Advances

    doi: 10.1126/sciadv.abf2467

    Antibody affinity maturation of human antibody response following SARS-CoV-2 infection in patients with COVID-19 and association with disease severity. ( A to D ) Polyclonal antibody affinity maturation to SARS-CoV-2 spike proteins was determined by SPR. Binding affinity of serially diluted after infection serum/plasma of each of the six patients with severe COVID-19 (in shades of red) and five mild patients (in shades of green) to prefusion spike (A), S1 domain (B), RBD (C), and S2 domain (D). All SPR experiments were performed twice, and data shown are average value of two experimental runs. Off-rate was calculated and shown only for samples that demonstrated antibody binding of 10 to 100 RU in SPR. ( E ) Antibody affinity (as measured by dissociation off-rate per second) against SARS-CoV-2 prefusion spike, S1, S2, and RBD for the final day samples from each of the severe (red) versus mild (green) COVID-19 patients. The statistical significances between the groups were determined using Kruskal-Wallis multiple comparisons test for severe patients (red) and mild patients (green). The differences were considered statistically significant with 95% confidence interval when P value was less than 0.05. * P
    Figure Legend Snippet: Antibody affinity maturation of human antibody response following SARS-CoV-2 infection in patients with COVID-19 and association with disease severity. ( A to D ) Polyclonal antibody affinity maturation to SARS-CoV-2 spike proteins was determined by SPR. Binding affinity of serially diluted after infection serum/plasma of each of the six patients with severe COVID-19 (in shades of red) and five mild patients (in shades of green) to prefusion spike (A), S1 domain (B), RBD (C), and S2 domain (D). All SPR experiments were performed twice, and data shown are average value of two experimental runs. Off-rate was calculated and shown only for samples that demonstrated antibody binding of 10 to 100 RU in SPR. ( E ) Antibody affinity (as measured by dissociation off-rate per second) against SARS-CoV-2 prefusion spike, S1, S2, and RBD for the final day samples from each of the severe (red) versus mild (green) COVID-19 patients. The statistical significances between the groups were determined using Kruskal-Wallis multiple comparisons test for severe patients (red) and mild patients (green). The differences were considered statistically significant with 95% confidence interval when P value was less than 0.05. * P

    Techniques Used: Infection, SPR Assay, Binding Assay

    Viral load, serum neutralizing titers, and cytokine analyses of patients with COVID-19 during hospitalization. ( A to K ) Viral load in upper respiratory tract of the 11 patients with COVID-19 at various time points as measured by RT–quantitative PCR (qPCR) (blue symbols). SARS-CoV-2–neutralizing antibody titers in serum/plasma for six severe (A to F; red symbols) and five mild (G to K; green symbols) COVID-19 patients at various time points as determined by PRNT50. ( L to O ) Cytokine levels (L, IL-6; M, IL-8; N, MCP-1; and O, MIP-1β) of fourfold diluted plasma/serum samples at various time points in the 11 patients with COVID-19 as analyzed via a Bio-Plex Pro Human Cytokine Panel 17-Plex assay. ( P ) AUC for the four cytokine/chemokines of the 11 patients with COVID-19. Limit of detection (LOD) for the assay is shown as dashed line. The statistical significances between the groups were determined using Kruskal-Wallis multiple comparisons test for AUC (AUC values) of severe patients (red) and mild patients (green). The differences were considered statistically significant with a 95% confidence interval when the P value was less than 0.05. * P
    Figure Legend Snippet: Viral load, serum neutralizing titers, and cytokine analyses of patients with COVID-19 during hospitalization. ( A to K ) Viral load in upper respiratory tract of the 11 patients with COVID-19 at various time points as measured by RT–quantitative PCR (qPCR) (blue symbols). SARS-CoV-2–neutralizing antibody titers in serum/plasma for six severe (A to F; red symbols) and five mild (G to K; green symbols) COVID-19 patients at various time points as determined by PRNT50. ( L to O ) Cytokine levels (L, IL-6; M, IL-8; N, MCP-1; and O, MIP-1β) of fourfold diluted plasma/serum samples at various time points in the 11 patients with COVID-19 as analyzed via a Bio-Plex Pro Human Cytokine Panel 17-Plex assay. ( P ) AUC for the four cytokine/chemokines of the 11 patients with COVID-19. Limit of detection (LOD) for the assay is shown as dashed line. The statistical significances between the groups were determined using Kruskal-Wallis multiple comparisons test for AUC (AUC values) of severe patients (red) and mild patients (green). The differences were considered statistically significant with a 95% confidence interval when the P value was less than 0.05. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Plex Assay

    IgM, IgG, and IgA antibody repertoires elicited in patient with severe COVID-19 who succumbed to SARS-CoV-2 infection. ( A ) Distribution of phage clones after affinity selection on post–SARS-CoV-2 infection samples. Number of IgM-, IgG-, and IgA-bound phage clones selected using SARS-CoV-2 spike GFPDL on polyclonal samples from days 6 (D6), 21 (D21), and 42 (D42) following symptom onset in patient with fatal COVID-19 (S-01). ( B to D ) IgM, IgG, and IgA antibody epitope repertoire recognized in the SARS-CoV-2–infected serum/plasma of deceased patient with COVID-19 (S-01) at different days after onset of symptoms and their alignment to the spike protein of SARS-CoV-2. CD, connector domain; CH, central helix; CT, cytoplasmic tail; FP, fusion peptide; HR, heptad repeat; SP, signal peptide. Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, is shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike on alignment. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value for IgM, IgG, and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data were performed in duplicate (two independent experiments by researcher in the laboratory, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis.
    Figure Legend Snippet: IgM, IgG, and IgA antibody repertoires elicited in patient with severe COVID-19 who succumbed to SARS-CoV-2 infection. ( A ) Distribution of phage clones after affinity selection on post–SARS-CoV-2 infection samples. Number of IgM-, IgG-, and IgA-bound phage clones selected using SARS-CoV-2 spike GFPDL on polyclonal samples from days 6 (D6), 21 (D21), and 42 (D42) following symptom onset in patient with fatal COVID-19 (S-01). ( B to D ) IgM, IgG, and IgA antibody epitope repertoire recognized in the SARS-CoV-2–infected serum/plasma of deceased patient with COVID-19 (S-01) at different days after onset of symptoms and their alignment to the spike protein of SARS-CoV-2. CD, connector domain; CH, central helix; CT, cytoplasmic tail; FP, fusion peptide; HR, heptad repeat; SP, signal peptide. Graphical distribution of representative clones with a frequency of ≥2, obtained after affinity selection, is shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike on alignment. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value for IgM, IgG, and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data were performed in duplicate (two independent experiments by researcher in the laboratory, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis.

    Techniques Used: Infection, Clone Assay, Selection, Sequencing, Isolation

    Evolution of IgM, IgG, and IgA antibody repertoire in patient with mild COVID-19. ( A ) Distribution of phage clones after affinity selection in serum following SARS-CoV-2 infection. Number of IgM, IgG, and IgA bound phage clones selected using SARS-CoV-2 spike GFPDL on polyclonal samples from days 5 (D5), 12 (D12), 15 (D15), and 25 (D25) following symptom onset in patient with mild COVID-19 (M-10). ( B to D ) IgM, IgG, and IgA antibody epitope repertoire recognized in the SARS-CoV-2–infected serum/plasma at different days after onset of symptoms 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, is shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike on alignment. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value for IgM, IgG, and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data were performed in duplicate (two independent experiments by researcher in the laboratory, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis.
    Figure Legend Snippet: Evolution of IgM, IgG, and IgA antibody repertoire in patient with mild COVID-19. ( A ) Distribution of phage clones after affinity selection in serum following SARS-CoV-2 infection. Number of IgM, IgG, and IgA bound phage clones selected using SARS-CoV-2 spike GFPDL on polyclonal samples from days 5 (D5), 12 (D12), 15 (D15), and 25 (D25) following symptom onset in patient with mild COVID-19 (M-10). ( B to D ) IgM, IgG, and IgA antibody epitope repertoire recognized in the SARS-CoV-2–infected serum/plasma at different days after onset of symptoms 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, is shown. The horizontal position and the length of the bars indicate the peptide sequence displayed on the selected phage clone to its homologous sequence in the SARS-CoV-2 spike on alignment. The thickness of each bar represents the frequency of repetitively isolated phage. Scale value for IgM, IgG, and IgA is shown enclosed in a red box beneath the respective alignments. The GFPDL affinity selection data were performed in duplicate (two independent experiments by researcher in the laboratory, who was blinded to sample identity), and similar number of phage clones and epitope repertoire was observed in both phage display analysis.

    Techniques Used: Clone Assay, Selection, Infection, Sequencing, Isolation

    Elucidation of IgM, IgG, and IgA antibody repertoire following SARS-CoV-2 infection in patient with mild COVID-19. ( A ) Distribution of phage clones after affinity selection with COVID-19 serum. Number of IgM, IgG, and IgA bound phage clones with days 1 (D1) and 14 (D14) serum following symptom onset of patient with mild COVID-19 (M-14). ( B to D ) SARS-CoV-2 IgM, IgG, and IgA epitope repertoire of the patient with mild COVID-19 (M-14). Graphical distribution of affinity-selected clones with frequency of ≥2 is shown. The horizontal position and length of bars indicate the peptide sequence displayed on selected phage clone to its homologous sequence in SARS-CoV-2 spike. Thickness of each bar represents frequency of repetitively isolated phage. Scale value for IgM, IgG, and IgA is shown in a red box beneath the respective alignments. ( E to G ) Antibody epitope profile following SARS-CoV-2 infection. Antigenic regions/sites within SARS-CoV-2 spike sequence (GenBank: MN908947.3) recognized by serum/plasma antibodies (based on data presented in Figs. 2 to 4 ). Antigenic sites shown in cyan were uniquely recognized by post–SARS-CoV-2 infection IgG (E), IgA (F), or IgM (G) only in patients with mild but not severe COVID-19. Antigenic sites shown in red were uniquely recognized by post–SARS-CoV-2 infection IgG (E), IgA (F), or IgM (G) antibodies only in patients with severe but not mild COVID-19. Epitopes of each protein are numbered in black.
    Figure Legend Snippet: Elucidation of IgM, IgG, and IgA antibody repertoire following SARS-CoV-2 infection in patient with mild COVID-19. ( A ) Distribution of phage clones after affinity selection with COVID-19 serum. Number of IgM, IgG, and IgA bound phage clones with days 1 (D1) and 14 (D14) serum following symptom onset of patient with mild COVID-19 (M-14). ( B to D ) SARS-CoV-2 IgM, IgG, and IgA epitope repertoire of the patient with mild COVID-19 (M-14). Graphical distribution of affinity-selected clones with frequency of ≥2 is shown. The horizontal position and length of bars indicate the peptide sequence displayed on selected phage clone to its homologous sequence in SARS-CoV-2 spike. Thickness of each bar represents frequency of repetitively isolated phage. Scale value for IgM, IgG, and IgA is shown in a red box beneath the respective alignments. ( E to G ) Antibody epitope profile following SARS-CoV-2 infection. Antigenic regions/sites within SARS-CoV-2 spike sequence (GenBank: MN908947.3) recognized by serum/plasma antibodies (based on data presented in Figs. 2 to 4 ). Antigenic sites shown in cyan were uniquely recognized by post–SARS-CoV-2 infection IgG (E), IgA (F), or IgM (G) only in patients with mild but not severe COVID-19. Antigenic sites shown in red were uniquely recognized by post–SARS-CoV-2 infection IgG (E), IgA (F), or IgM (G) antibodies only in patients with severe but not mild COVID-19. Epitopes of each protein are numbered in black.

    Techniques Used: Infection, Clone Assay, Selection, Sequencing, Isolation

    SPR-based analysis of human antibody response following SARS-CoV-2 infection. Serial dilutions of each serum/plasma sample collected at different time points from patients with COVID-19 were analyzed for antibody binding to SARS-CoV-2 spike and subdomains (figs. S7 and S8). ( A to D ) Total antibody binding is represented in SPR RU for six patients with severe COVID-19 (in shades of red) and five mild patients (in shades of green) for binding to prefusion spike (A), S1 domain (B), RBD (C), and S2 domain (D). Total antibody binding shown is observed maximum RU for 10-fold diluted serum/plasma sample. ( E ) Antibody isotype of SARS-CoV-2 prefusion spike–binding antibodies. The isotype composition of serum/plasma antibodies bound to prefusion spike. The RU for each anti-spike protein antibody isotype was divided by total RU for all antibody isotypes combined to calculate percentage of each antibody isotype for individual serum/plasma sample. ( F ) Mean percentage of IgM, IgG, and IgA antibody isotype bound to SARS-CoV-2 prefusion spike is shown for patients with severe (red) versus mild (green) COVID-19. The statistical significances between groups were determined using Kruskal-Wallis multiple comparisons test for patients with severe (red) versus mild (green) COVID-19. The differences were considered statistically significant with 95% confidence interval when P value was less than 0.05. ** P
    Figure Legend Snippet: SPR-based analysis of human antibody response following SARS-CoV-2 infection. Serial dilutions of each serum/plasma sample collected at different time points from patients with COVID-19 were analyzed for antibody binding to SARS-CoV-2 spike and subdomains (figs. S7 and S8). ( A to D ) Total antibody binding is represented in SPR RU for six patients with severe COVID-19 (in shades of red) and five mild patients (in shades of green) for binding to prefusion spike (A), S1 domain (B), RBD (C), and S2 domain (D). Total antibody binding shown is observed maximum RU for 10-fold diluted serum/plasma sample. ( E ) Antibody isotype of SARS-CoV-2 prefusion spike–binding antibodies. The isotype composition of serum/plasma antibodies bound to prefusion spike. The RU for each anti-spike protein antibody isotype was divided by total RU for all antibody isotypes combined to calculate percentage of each antibody isotype for individual serum/plasma sample. ( F ) Mean percentage of IgM, IgG, and IgA antibody isotype bound to SARS-CoV-2 prefusion spike is shown for patients with severe (red) versus mild (green) COVID-19. The statistical significances between groups were determined using Kruskal-Wallis multiple comparisons test for patients with severe (red) versus mild (green) COVID-19. The differences were considered statistically significant with 95% confidence interval when P value was less than 0.05. ** P

    Techniques Used: SPR Assay, Infection, Binding Assay

    15) Product Images from "Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies"

    Article Title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422550

    The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and
    Figure Legend Snippet: The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and

    Techniques Used:

    Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .
    Figure Legend Snippet: Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .

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

    The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.
    Figure Legend Snippet: The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.

    Techniques Used: Sequencing, Mutagenesis

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    Binding Assay:

    Article Title: Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2
    Article Snippet: .. Binding Assays SARS-CoV-2 S1 and RBD (cat. no. 40591-V08H and 40592-V08H), SARS-CoV S1+S2 (cat. no. 40634-V08B), HCoV-NL63 S1 (cat. no. 40600-V08H; all with His tag), and ACE2-Fc (cat. no. 10108-H05H) used in the binding assay were obtained from SinoBiological (Wayne, PA, USA). .. The TNF-R1:Fc receptor (cat. no. ALX-522-013-C050) and its FLAG-tagged TNF-α ligand (cat. no. ALX-522-008-C050) were obtained from Enzo Life Sciences (San Diego, CA, USA).

    Multiplex Assay:

    Article Title: SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence
    Article Snippet: Assay Procedure The steps in assay validation were similar to recently developed bead-based multiplex immunoassays for CMV, EBV, and RSV, with minor modifications as described below [ , ]. .. For the multiplex bead-based immune assay the following antigens obtained from Sino Biological were used: SARS-CoV-2 monomeric spike S1 (40591-V08H), RBD (40592-V08B), and nucleoprotein (N) (40588-V08B). ..

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    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
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    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

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

    doi: 10.1002/smtd.202001031

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

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

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

    Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) ( a ). BALB/c mice ( n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S ΔTM , pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies ( b ). All data are represented as individual values. ** p

    Journal: Vaccines

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    doi: 10.3390/vaccines9040307

    Figure Lengend Snippet: Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) ( a ). BALB/c mice ( n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S ΔTM , pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies ( b ). All data are represented as individual values. ** p

    Article Snippet: Ninety-six-well immunosorbent plates (NUNC) were coated with 1 μg/mL recombinant SARS-CoV-2 S1+S2 ECD protein (Sino Biological 40589-V08B1) and S1 protein (Sino Biological 40591-V08H) in PBS (phosphate-buffered saline) overnight at 4 °C.

    Techniques: Expressing, Mouse Assay, Plasmid Preparation

    Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A , Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B , Laboratory-confirmed viral infections (see Supplementary Table 2 ) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.

    Journal: The Journal of Infectious Diseases

    Article Title: SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

    doi: 10.1093/infdis/jiaa479

    Figure Lengend Snippet: Discrimination of COVID-19 patients with varying severity from a cross-sectional population panel and ILI patients. A , Individuals from the cross-sectional panel aged 3–90 years (n = 224), ILI patients with noncoronavirus (n = 75), and non-SARS-CoV-2 seasonal coronavirus-infected ILI patients (n = 109) were compared to hospitalized and nonhospitalized COVID-19 patients. Median concentration and 95% confidence intervals and statistical results (adjusted P values of Tukey multiple comparison) between the groups are shown. B , Laboratory-confirmed viral infections (see Supplementary Table 2 ) and concentration data of ILI patients are shown to confirm that the assay discriminates SARS-CoV-2–specific antibodies from antibodies induced by various laboratory-confirmed viral infections. Abbreviations: AU, arbitrary unit; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleoprotein; non-HCoV, noncoronavirus; RBD, receptor binding domain; RSV, respiratory syncytial virus; S1, spike protein subunit 1.

    Article Snippet: For the multiplex bead-based immune assay the following antigens obtained from Sino Biological were used: SARS-CoV-2 monomeric spike S1 (40591-V08H), RBD (40592-V08B), and nucleoprotein (N) (40588-V08B).

    Techniques: Infection, Concentration Assay, Binding Assay

    Few antigen-specific ASC induced in circulation after the second vaccine dose in SARS-CoV-2-experienced adults. ( A and B ). Antibody-secreting cell (ASC) ELISpots for a SARS-CoV-2-naive ( A ) or SARS-CoV-2-experienced ( B ) adult one week after each dose of vaccine. ( C to F ) Summary statistics for ELISpot assays. For each panel, S1 (left), S2 (middle), or RBD (right) antigens for IgG or IgA are represented, at one week after first dose ( C and D ) or second dose ( E and F ). Nominal P values from Wilcoxon tests. ( G to I ) ELISpot results for SARS-CoV-2-naive (left) or SARS-CoV-2-experienced (right) adults for S1 ( G ), S2 ( H ), or RBD ( I ). Connected lines indicate repeated measurements from the same participants. Nominal P values from paired t-tests. ( J and K ) Kendall correlations for ELISpot results one week after the first vaccination ( J ) or one week after the second vaccination ( K ). Correlations shown for comparisons with nominal P values

    Journal: medRxiv

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    doi: 10.1101/2021.02.07.21251311

    Figure Lengend Snippet: Few antigen-specific ASC induced in circulation after the second vaccine dose in SARS-CoV-2-experienced adults. ( A and B ). Antibody-secreting cell (ASC) ELISpots for a SARS-CoV-2-naive ( A ) or SARS-CoV-2-experienced ( B ) adult one week after each dose of vaccine. ( C to F ) Summary statistics for ELISpot assays. For each panel, S1 (left), S2 (middle), or RBD (right) antigens for IgG or IgA are represented, at one week after first dose ( C and D ) or second dose ( E and F ). Nominal P values from Wilcoxon tests. ( G to I ) ELISpot results for SARS-CoV-2-naive (left) or SARS-CoV-2-experienced (right) adults for S1 ( G ), S2 ( H ), or RBD ( I ). Connected lines indicate repeated measurements from the same participants. Nominal P values from paired t-tests. ( J and K ) Kendall correlations for ELISpot results one week after the first vaccination ( J ) or one week after the second vaccination ( K ). Correlations shown for comparisons with nominal P values

    Article Snippet: Ninety-six well ELISpot filter plates (Millipore, MSHAN4B50) were coated overnight with 2 µg/mL recombinant S1, S2, or RBD (Sino Biological Inc., 40591-V08H, 40590-V08B, and 40592-V08H), or 10 µg/mL of goat anti-human IgG/A/M capture antibody (Jackson ImmunoResearch Laboratory Inc., 109-005-064).

    Techniques: Enzyme-linked Immunospot

    SARS-CoV-2-experienced individuals’ robust anti-S1 binding and neutralizing antibodies responses after vaccination. ( A ) Anti-S1 IgG serum antibody titers over time measured in days relative to the first vaccination. ( B ) Anti-S1 IgG titer one week after second vaccination for each cohort ( P =0.06; Wilcoxon test). ( C ) Anti-nucleocapsid IgG serum antibody titers. ( D ) Correlation between fold-change in anti-S1 IgG serum antibody titers, assessed as one week after vaccination compared to baseline, compared to the baseline anti-S1 IgG serum antibody titers, for SARS-CoV-2-experienced adults. ( E ) Example of neutralizing antibody assay shown for the same SARS-CoV-2-experienced participant longitudinally. ( F ) Neutralizing antibody titers one week after the second vaccination ( P =0.19; Wilcoxon test).

    Journal: medRxiv

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    doi: 10.1101/2021.02.07.21251311

    Figure Lengend Snippet: SARS-CoV-2-experienced individuals’ robust anti-S1 binding and neutralizing antibodies responses after vaccination. ( A ) Anti-S1 IgG serum antibody titers over time measured in days relative to the first vaccination. ( B ) Anti-S1 IgG titer one week after second vaccination for each cohort ( P =0.06; Wilcoxon test). ( C ) Anti-nucleocapsid IgG serum antibody titers. ( D ) Correlation between fold-change in anti-S1 IgG serum antibody titers, assessed as one week after vaccination compared to baseline, compared to the baseline anti-S1 IgG serum antibody titers, for SARS-CoV-2-experienced adults. ( E ) Example of neutralizing antibody assay shown for the same SARS-CoV-2-experienced participant longitudinally. ( F ) Neutralizing antibody titers one week after the second vaccination ( P =0.19; Wilcoxon test).

    Article Snippet: Ninety-six well ELISpot filter plates (Millipore, MSHAN4B50) were coated overnight with 2 µg/mL recombinant S1, S2, or RBD (Sino Biological Inc., 40591-V08H, 40590-V08B, and 40592-V08H), or 10 µg/mL of goat anti-human IgG/A/M capture antibody (Jackson ImmunoResearch Laboratory Inc., 109-005-064).

    Techniques: Binding Assay

    Antibody responses differ based on prior history of COVID-19. ( A ) Anti-S1 IgG antibody titers were assessed for SARS-CoV-2 experienced (purple) and SARS-CoV-2-naive (yellow) adults. Connected lines indicate repeated measurements of the same participants. * P

    Journal: medRxiv

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    doi: 10.1101/2021.02.07.21251311

    Figure Lengend Snippet: Antibody responses differ based on prior history of COVID-19. ( A ) Anti-S1 IgG antibody titers were assessed for SARS-CoV-2 experienced (purple) and SARS-CoV-2-naive (yellow) adults. Connected lines indicate repeated measurements of the same participants. * P

    Article Snippet: Ninety-six well ELISpot filter plates (Millipore, MSHAN4B50) were coated overnight with 2 µg/mL recombinant S1, S2, or RBD (Sino Biological Inc., 40591-V08H, 40590-V08B, and 40592-V08H), or 10 µg/mL of goat anti-human IgG/A/M capture antibody (Jackson ImmunoResearch Laboratory Inc., 109-005-064).

    Techniques:

    CD4 and CD8 T cell responses and gating strategy. ( A ) Study participant timeline relative to first vaccination. ( B ) Gating scheme for T and B cell populations. ( C ) Non-naive CD8 shown in tSNE projection for SARS-CoV-2-naive (upper) or SARS-CoV-2-experienced (lower) participants. Heatmap shows expression of Ki67. Circled area indicates region corresponding to Cluster 12. ( D ) Non-naive CD8 underwent Phenograph clustering. Protein expression for each cluster for SARS-CoV-2-experienced adults shown for samples taken one week after the second vaccination. ( E and F ) Ki67+CD38+ expression in CD8 T cells by cohort over time measured in days, relative to the individual’s first ( E ) or second ( L ) vaccination. ( G ) Example for Ki67+CD38+ CD8 T cell expression of granzyme B in a SARS-CoV-2-experienced individual. ( H ) Summary data for Ki67+CD38+ CD8 T cell expression of granzyme B. P -values from one-way ANOVA with Tukey’s post test. ( I ) Non-naive CD4 T cells from all samples were merged for tSNE projection. Colors indicate Phenograph clustering. ( J and K ) Protein expression for Phenograph clusters for non-naive CD4 T cells shown for samples at one week following second vaccination in SARS-CoV-2-naive ( J ) or SARS-CoV-2-experienced ( K ) participants. ( L and M ) CD4 T cells shown for expression of Ki67 and CD38 after vaccination over time measured in days, relative to the individual’s first ( L ) or second ( M ) vaccinations. ( N ) Correlation between Ki67+CD38+ CD4 T cells and age one after either first vaccination (left) or second vaccination (right).

    Journal: medRxiv

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    doi: 10.1101/2021.02.07.21251311

    Figure Lengend Snippet: CD4 and CD8 T cell responses and gating strategy. ( A ) Study participant timeline relative to first vaccination. ( B ) Gating scheme for T and B cell populations. ( C ) Non-naive CD8 shown in tSNE projection for SARS-CoV-2-naive (upper) or SARS-CoV-2-experienced (lower) participants. Heatmap shows expression of Ki67. Circled area indicates region corresponding to Cluster 12. ( D ) Non-naive CD8 underwent Phenograph clustering. Protein expression for each cluster for SARS-CoV-2-experienced adults shown for samples taken one week after the second vaccination. ( E and F ) Ki67+CD38+ expression in CD8 T cells by cohort over time measured in days, relative to the individual’s first ( E ) or second ( L ) vaccination. ( G ) Example for Ki67+CD38+ CD8 T cell expression of granzyme B in a SARS-CoV-2-experienced individual. ( H ) Summary data for Ki67+CD38+ CD8 T cell expression of granzyme B. P -values from one-way ANOVA with Tukey’s post test. ( I ) Non-naive CD4 T cells from all samples were merged for tSNE projection. Colors indicate Phenograph clustering. ( J and K ) Protein expression for Phenograph clusters for non-naive CD4 T cells shown for samples at one week following second vaccination in SARS-CoV-2-naive ( J ) or SARS-CoV-2-experienced ( K ) participants. ( L and M ) CD4 T cells shown for expression of Ki67 and CD38 after vaccination over time measured in days, relative to the individual’s first ( L ) or second ( M ) vaccinations. ( N ) Correlation between Ki67+CD38+ CD4 T cells and age one after either first vaccination (left) or second vaccination (right).

    Article Snippet: Ninety-six well ELISpot filter plates (Millipore, MSHAN4B50) were coated overnight with 2 µg/mL recombinant S1, S2, or RBD (Sino Biological Inc., 40591-V08H, 40590-V08B, and 40592-V08H), or 10 µg/mL of goat anti-human IgG/A/M capture antibody (Jackson ImmunoResearch Laboratory Inc., 109-005-064).

    Techniques: Expressing

    mRNA vaccination induces CD4 and CD8 T cell responses. ( A ) Study schematic. ( B ) Non-naive CD8 T cells from all participants were colored using Phenograph clusters and projected using tSNE. Circled region indicates cluster 12. ( C ) Phenograph cluster abundance for non-naive CD8 T cells was compared using edgeR for all participants before and after the second vaccination. ( D ) Heatmap for non-naive CD8 T cell cluster protein expression for SARS-CoV-2-naive participants after the second vaccination. ( E ) Example of CD8 T cell expression of Ki67 and CD38. ( F ) Summary data for Ki67+CD38+ expression in CD8 T cells by cohort. * P

    Journal: medRxiv

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    doi: 10.1101/2021.02.07.21251311

    Figure Lengend Snippet: mRNA vaccination induces CD4 and CD8 T cell responses. ( A ) Study schematic. ( B ) Non-naive CD8 T cells from all participants were colored using Phenograph clusters and projected using tSNE. Circled region indicates cluster 12. ( C ) Phenograph cluster abundance for non-naive CD8 T cells was compared using edgeR for all participants before and after the second vaccination. ( D ) Heatmap for non-naive CD8 T cell cluster protein expression for SARS-CoV-2-naive participants after the second vaccination. ( E ) Example of CD8 T cell expression of Ki67 and CD38. ( F ) Summary data for Ki67+CD38+ expression in CD8 T cells by cohort. * P

    Article Snippet: Ninety-six well ELISpot filter plates (Millipore, MSHAN4B50) were coated overnight with 2 µg/mL recombinant S1, S2, or RBD (Sino Biological Inc., 40591-V08H, 40590-V08B, and 40592-V08H), or 10 µg/mL of goat anti-human IgG/A/M capture antibody (Jackson ImmunoResearch Laboratory Inc., 109-005-064).

    Techniques: Expressing

    Poor IgG and IgA ASC responses to second dose in SARS-CoV-2 experienced participants. ( A and B ) Antibody-secreting cell (ASC) ELISpots shown for IgG, IgA, and IgM-producing cells reacting to RBD, S1, or S2 antigens, or total secreted antibody controls.. ( C ) IgM-producing ASC in circulation quantified one week after first vaccination (top) or one week after second vaccination (bottom). Nominal P values from Wilcoxon tests. ( D ) ASC frequencies shown over time measured in days relative to the first vaccination (top) or second vaccination (bottom) for S1, S2, or RBD antigens. ( E and F ) Correlation shown for both cohorts for S1-reactive IgG ASC ( E ) or S2-reactive IgG ASC ( F ) compared to RBD-reactive IgG ASC one week after second vaccination. ( G and H ) Kendall correlation shown for SARS-CoV-2-specific frequencies for SARS-CoV-2-naive or SARS-CoV-2-experienced adults one week after first ( G ) or one week after second ( H ) vaccination. Heatmap colored by Kendall’s tau statistic. Boxes with symbols indicate nominal P value > 0.05.

    Journal: medRxiv

    Article Title: Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals

    doi: 10.1101/2021.02.07.21251311

    Figure Lengend Snippet: Poor IgG and IgA ASC responses to second dose in SARS-CoV-2 experienced participants. ( A and B ) Antibody-secreting cell (ASC) ELISpots shown for IgG, IgA, and IgM-producing cells reacting to RBD, S1, or S2 antigens, or total secreted antibody controls.. ( C ) IgM-producing ASC in circulation quantified one week after first vaccination (top) or one week after second vaccination (bottom). Nominal P values from Wilcoxon tests. ( D ) ASC frequencies shown over time measured in days relative to the first vaccination (top) or second vaccination (bottom) for S1, S2, or RBD antigens. ( E and F ) Correlation shown for both cohorts for S1-reactive IgG ASC ( E ) or S2-reactive IgG ASC ( F ) compared to RBD-reactive IgG ASC one week after second vaccination. ( G and H ) Kendall correlation shown for SARS-CoV-2-specific frequencies for SARS-CoV-2-naive or SARS-CoV-2-experienced adults one week after first ( G ) or one week after second ( H ) vaccination. Heatmap colored by Kendall’s tau statistic. Boxes with symbols indicate nominal P value > 0.05.

    Article Snippet: Ninety-six well ELISpot filter plates (Millipore, MSHAN4B50) were coated overnight with 2 µg/mL recombinant S1, S2, or RBD (Sino Biological Inc., 40591-V08H, 40590-V08B, and 40592-V08H), or 10 µg/mL of goat anti-human IgG/A/M capture antibody (Jackson ImmunoResearch Laboratory Inc., 109-005-064).

    Techniques: