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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    Comparison of seroconversion in patients with <t>COVID-19</t> and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with <t>SARS-CoV-2</t> and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS <t>2017–2019</t> (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
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    1) Product Images from "Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity"

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

    Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.
    Figure Legend Snippet: Comparison of seroconversion in patients with COVID-19 and healthy individuals. ( A ) ELISA with S-RBD protein coating and 1:100 dilution of repeated serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 88 (from 21 patients); HS 2017–2019 (white), n = 104; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (left inset) and 2020 (right inset). ( B ) ELISA with N-protein coating and 1:100 dilution of the first and last serum samples of patients with SARS-CoV-2 and healthy individuals. Absorbance normalized to the respective no antigen control for each sample at 450 nm reported. SARS-CoV-2 (blue), n = 37 (from 21 patients); HS 2017–2019 (white), n = 103; HS 2020 (white), n = 308. Arrows list consecutive serum samples evaluated for each case. Inset graphs depict the data separated based on healthy serum collected from 2017 to 2019 (top inset) and 2020 (bottom inset). ( C ) Pie charts depicting percentage of samples positive for indicated antigens. SARS-CoV-2, n = 21; HS 2017–2019, n = 103; HS 2020, n = 308; non–COVID-19 samples (NCSs), n = 45; HIV, n = 7; all, n = 484.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P
    Figure Legend Snippet: Detection of serum binding antibodies against SARS-CoV-2 proteins in patients with PCR-confirmed COVID-19 and healthy samples. ( A ) Timeline of COVID-19 diagnosis/ICU admittance, serum sample collection, and convalescent plasma (CP) administration. Time 0 is defined as day of COVID-19 diagnosis (PCR positive for SARS-CoV-2) and ICU admittance. Blood collections are denoted in gray and CP administration is denoted in pink. Patients were stratified based on current status (recovered, hospitalized, or deceased). Patient 29 from our cohort had symptoms but was PCR negative for SARS-CoV-2; this sample was not included in figures since there was no proof of disease. ( B ) Schematic of SARS-CoV-2 viral structure (top panel) and antigens assayed (bottom panel). S-protein, light orange; envelope protein, yellow; membrane glycoprotein, dark orange; RNA, blue; N-protein, green. Absorbance normalized to the respective no antigen control for each sample at 450 nm plotted for S-RBD (left panel), and N-protein (right panel), antigen coating with the most recent (or only) SARS-CoV-2 samples not treated with CP ( n = 21) and healthy samples collected in 2017–2019 (HS 2017–2019, n = 104 for S-RBD, n = 103 for N-protein) and 2020 (HS 2020, n = 308). Data are presented with each dot representing the mean normalized absorbance for a given serum sample; the red bar depicts the median ± interquartile range of all samples. HS, healthy sample; NC (line), negative control cutoff (see Methods). Kruskal-Wallis with Dunn’s multiple-comparisons test performed. **** P

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Negative Control

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

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

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00301-9

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

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

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

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

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

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    3) Product Images from "The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection"

    Article Title: The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection

    Journal: bioRxiv

    doi: 10.1101/2021.03.22.436522

    Halofuginone Inhibits infection and replication of authentic SARS-CoV-2. a-b, Authentic SARS-CoV-2 virus infection of Huh7.5 cells treated with Halofuginone. Huh7.5 cells treated with halofuginone pre- or post-infection, or both pre- and post-infection with authentic SARS-CoV-2 virus. Viral titers ( a ) and quantification of viral RNA ( b ) in the infected cells is shown. c, Immunofluorescent quantification of viral nucleocapsid (red) protein in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus (nuclei = green). d, Authentic SARS-CoV-2 virus infection of Vero E6 cells treated with Halofuginone and Remdesivir as measured in flow cytometry. e, Quantification of plaque formation and viral RNA in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus. f, Rescue experiment of the effect of halofuginone treatment on SARS-CoV-2 infection using excess proline. g , Treatment of Vero E6 cells with halofuginone and enantiomers and their effect on SARS-CoV-2 infection. h, Treatment of Vero E6 cells with modulators of the PRS pathway and their effect on SARS-CoV-2 infection. i, Treatment of Vero E6 cells with modulators of the ISR pathway and their effect on SARS-CoV-2 infection. j, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins and viral SARS-CoV-2 (SARS2), SARS-CoV-1 (SARS1), MERS-CoV (MERS), HCoV-229E and HCoV-LN63 proteins( ## p ≤ 0.01 collagen s vs. all other protein classes). k, Treatment of Vero E6 cells with AARS inhibitors and their effect on SARS-CoV-2 infection. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001, ## : p ≤ 0.01.
    Figure Legend Snippet: Halofuginone Inhibits infection and replication of authentic SARS-CoV-2. a-b, Authentic SARS-CoV-2 virus infection of Huh7.5 cells treated with Halofuginone. Huh7.5 cells treated with halofuginone pre- or post-infection, or both pre- and post-infection with authentic SARS-CoV-2 virus. Viral titers ( a ) and quantification of viral RNA ( b ) in the infected cells is shown. c, Immunofluorescent quantification of viral nucleocapsid (red) protein in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus (nuclei = green). d, Authentic SARS-CoV-2 virus infection of Vero E6 cells treated with Halofuginone and Remdesivir as measured in flow cytometry. e, Quantification of plaque formation and viral RNA in Vero E6 cells treated with Halofuginone and Remdesivir and infected with authentic SARS-CoV-2 virus. f, Rescue experiment of the effect of halofuginone treatment on SARS-CoV-2 infection using excess proline. g , Treatment of Vero E6 cells with halofuginone and enantiomers and their effect on SARS-CoV-2 infection. h, Treatment of Vero E6 cells with modulators of the PRS pathway and their effect on SARS-CoV-2 infection. i, Treatment of Vero E6 cells with modulators of the ISR pathway and their effect on SARS-CoV-2 infection. j, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins and viral SARS-CoV-2 (SARS2), SARS-CoV-1 (SARS1), MERS-CoV (MERS), HCoV-229E and HCoV-LN63 proteins( ## p ≤ 0.01 collagen s vs. all other protein classes). k, Treatment of Vero E6 cells with AARS inhibitors and their effect on SARS-CoV-2 infection. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001, ## : p ≤ 0.01.

    Techniques Used: Infection, Flow Cytometry

    Halofuginone Inhibition of heparan sulfate presentation and Spike protein binding is not dependent of the integrated stress response. a, Chemical structure of halofuginone and negative control compound MAZ1310. Figure shows the interaction of halofuginone with the human prolyl-tRNA synthetase (PRS) active site, as resolved by X-ray crystallography (PDB: 4K88) 48 . b, Chemical structure of ProSA. Graphic shows the interaction of ProSA with human PRS (PDB: 5V58) 49 . c, Schematic representation of the mediators of the integrated stress response (ISR). c-d, Treatment of Hep3B cells with modulators of the PRS pathway at 0.5 µM (ProSA at 5 µM) and its effect on ( c ) HS presentation as measured by anti-HS mAb 10E4 binding and ( d ) spike RBD binding ( e ) by flow cytometry. Binding is represented as relative to non-treated control. f, Treatment of Hep3B cells with modulators of the halofuginone with or without 4mM proline and its effect on spike RBD binding by flow cytometry. Binding is represented as relative to non-treated control. g, Treatment of Hep3B cells with modulators of the ISR and its effect on HS presentation as measured by anti-HS mAb 10E4 binding in flow cytometry. h, Treatment of Hep3B cells with modulators of the ISR and its effect on SARS-CoV-2 recombinant RBD protein binding in flow cytometry. Binding is represented as relative to non-treated control. i, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins, heparan sulfate (HS) biosynthetic proteins, heparan sulfate proteoglycans (HSPG) and viral host factor proteins ( ## p ≤ 0.01 collagens vs. all other protein classes). Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001; # : p ≤ 0.05, ## : p ≤ 0.01).
    Figure Legend Snippet: Halofuginone Inhibition of heparan sulfate presentation and Spike protein binding is not dependent of the integrated stress response. a, Chemical structure of halofuginone and negative control compound MAZ1310. Figure shows the interaction of halofuginone with the human prolyl-tRNA synthetase (PRS) active site, as resolved by X-ray crystallography (PDB: 4K88) 48 . b, Chemical structure of ProSA. Graphic shows the interaction of ProSA with human PRS (PDB: 5V58) 49 . c, Schematic representation of the mediators of the integrated stress response (ISR). c-d, Treatment of Hep3B cells with modulators of the PRS pathway at 0.5 µM (ProSA at 5 µM) and its effect on ( c ) HS presentation as measured by anti-HS mAb 10E4 binding and ( d ) spike RBD binding ( e ) by flow cytometry. Binding is represented as relative to non-treated control. f, Treatment of Hep3B cells with modulators of the halofuginone with or without 4mM proline and its effect on spike RBD binding by flow cytometry. Binding is represented as relative to non-treated control. g, Treatment of Hep3B cells with modulators of the ISR and its effect on HS presentation as measured by anti-HS mAb 10E4 binding in flow cytometry. h, Treatment of Hep3B cells with modulators of the ISR and its effect on SARS-CoV-2 recombinant RBD protein binding in flow cytometry. Binding is represented as relative to non-treated control. i, The distribution of proline distribution and density depicted as a proline distribution score for collagens, cholesterol biosynthetic proteins, lysosomal proteins, heparan sulfate (HS) biosynthetic proteins, heparan sulfate proteoglycans (HSPG) and viral host factor proteins ( ## p ≤ 0.01 collagens vs. all other protein classes). Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001; # : p ≤ 0.05, ## : p ≤ 0.01).

    Techniques Used: Inhibition, Protein Binding, Negative Control, Binding Assay, Flow Cytometry, Recombinant

    Screen of epigenetic and translational regulatory compounds identify Halofuginone as a potent inhibitor of SARS-CoV-2 Spike HS dependent cellular adhesion. a, Hep3B cells were treated with a library of epigenetic and translational regulatory compounds or heparin lyase (HSase) as a positive control and tested for their interaction with recombinant SARS-CoV-2 RBD protein. b-e, Titration of halofuginone on Hep3B cells ( b ), Vero E6 ( c ), Calu-3 ( d ) and Cacu-2 ( e ) cells (n= 2-4 replicates per condition), and its effect on binding of recombinant SARS-CoV-2 RBD protein Heparin Lyase treatment is included as control for HS dependent adhesion. f-g, effect of halofuginone treatment on the infection of SARS-CoV-2 spike protein pseudotyped virus in Hep3B ( f ) and Vero E6 ( g ) cells (n= 3-4 replicates per condition). h , Authentic SARS-CoV-2 virus infection of Hep3B cells treated with halofuginone. i, Infection of primary human bronchial epithelial cells, grown at an air-liquid interface, treated with halofuginone (Red and black colors represent the two different primary human bronchial epithelial cells isolates used). j , Relative cell viability of primary human bronchial epithelial cells. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).
    Figure Legend Snippet: Screen of epigenetic and translational regulatory compounds identify Halofuginone as a potent inhibitor of SARS-CoV-2 Spike HS dependent cellular adhesion. a, Hep3B cells were treated with a library of epigenetic and translational regulatory compounds or heparin lyase (HSase) as a positive control and tested for their interaction with recombinant SARS-CoV-2 RBD protein. b-e, Titration of halofuginone on Hep3B cells ( b ), Vero E6 ( c ), Calu-3 ( d ) and Cacu-2 ( e ) cells (n= 2-4 replicates per condition), and its effect on binding of recombinant SARS-CoV-2 RBD protein Heparin Lyase treatment is included as control for HS dependent adhesion. f-g, effect of halofuginone treatment on the infection of SARS-CoV-2 spike protein pseudotyped virus in Hep3B ( f ) and Vero E6 ( g ) cells (n= 3-4 replicates per condition). h , Authentic SARS-CoV-2 virus infection of Hep3B cells treated with halofuginone. i, Infection of primary human bronchial epithelial cells, grown at an air-liquid interface, treated with halofuginone (Red and black colors represent the two different primary human bronchial epithelial cells isolates used). j , Relative cell viability of primary human bronchial epithelial cells. Data shown as mean ± S.D. Statistics performed by 1-way ANOVA and uncorrected Fisher’s LSD test (ns: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001).

    Techniques Used: Positive Control, Recombinant, Titration, Binding Assay, Infection

    4) Product Images from "A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)"

    Article Title: A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112715

    Laboratory confirmation of ACE2-based LFIA using clinical samples a) Schematic diagram of COVID-19 test using ACE2-based LFIA. A nasopharyngeal swab from the COVID-19 patient is placed into the UTM. 50 μL of UTM containing the SARS-CoV-2 is mixed with running buffer in a 1:1 (v/v) ratio, and 100 μL of mixed solution is loaded into the LFIA device. After 20 min, the line intensity of the LFIA strip is semi-quantified by the portable analyzer. b) Results of ACE2-based LFA for the detection sensitivity of cultured SARS-CoV-2. Serially diluted virus concentrates (concentration range: 1.07 × 10 8 copies/mL to 5.35 × 10 6 copies/mL) were tested. After 20 min, the LFIA strip was taken with a smartphone and scanned with an image analyzer. The line intensities of the test and control lines were converted to peak histograms. Also, the intensity of the test lines was measured by a portable line analyzer (I L : line intensity of test line). Furthermore, human coronavirus (OC43) was tested as a negative control. c) Bar graph of intensities for test lines measured by the portable analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (0 copies/mL of SARS-CoV-2) plus three times the standard deviation. d) Laboratory confirmation of ACE2-based LFIA compared to the RT-qPCR using clinical samples. i) Nasopharyngeal swab samples of COVID-19 patients (n = 4) and healthy subjects (n = 4) were tested both ACE2-based LFIA and RT-qPCR. Sensitivity was determined by the number of true positive samples divided by the number of positive samples tested. Moreover, specificity was determined by the number of true negative samples divide by the number of negative samples tested. ii) RT-qPCR results for the detection of the SARS-CoV-2 specific gene (Env gene). C t value and their correspondent viral load in the clinical samples were evaluated. e) Results of ACE2-based LFIA on laboratory confirmation using clinical COVID-19 patient samples. Twenty minutes after sample loading, the test line intensities of the LFIA strips were measured with a portable line analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (healthy control) plus three times the standard deviation.
    Figure Legend Snippet: Laboratory confirmation of ACE2-based LFIA using clinical samples a) Schematic diagram of COVID-19 test using ACE2-based LFIA. A nasopharyngeal swab from the COVID-19 patient is placed into the UTM. 50 μL of UTM containing the SARS-CoV-2 is mixed with running buffer in a 1:1 (v/v) ratio, and 100 μL of mixed solution is loaded into the LFIA device. After 20 min, the line intensity of the LFIA strip is semi-quantified by the portable analyzer. b) Results of ACE2-based LFA for the detection sensitivity of cultured SARS-CoV-2. Serially diluted virus concentrates (concentration range: 1.07 × 10 8 copies/mL to 5.35 × 10 6 copies/mL) were tested. After 20 min, the LFIA strip was taken with a smartphone and scanned with an image analyzer. The line intensities of the test and control lines were converted to peak histograms. Also, the intensity of the test lines was measured by a portable line analyzer (I L : line intensity of test line). Furthermore, human coronavirus (OC43) was tested as a negative control. c) Bar graph of intensities for test lines measured by the portable analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (0 copies/mL of SARS-CoV-2) plus three times the standard deviation. d) Laboratory confirmation of ACE2-based LFIA compared to the RT-qPCR using clinical samples. i) Nasopharyngeal swab samples of COVID-19 patients (n = 4) and healthy subjects (n = 4) were tested both ACE2-based LFIA and RT-qPCR. Sensitivity was determined by the number of true positive samples divided by the number of positive samples tested. Moreover, specificity was determined by the number of true negative samples divide by the number of negative samples tested. ii) RT-qPCR results for the detection of the SARS-CoV-2 specific gene (Env gene). C t value and their correspondent viral load in the clinical samples were evaluated. e) Results of ACE2-based LFIA on laboratory confirmation using clinical COVID-19 patient samples. Twenty minutes after sample loading, the test line intensities of the LFIA strips were measured with a portable line analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (healthy control) plus three times the standard deviation.

    Techniques Used: Stripping Membranes, Cell Culture, Concentration Assay, Negative Control, Standard Deviation, Quantitative RT-PCR

    5) Product Images from "SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques"

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    Journal: bioRxiv

    doi: 10.1101/2021.04.05.438479

    SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    Figure Legend Snippet: SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).

    Techniques Used: Functional Assay, Blocking Assay, Neutralization

    Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.
    Figure Legend Snippet: Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.

    Techniques Used: Staining

    Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.
    Figure Legend Snippet: Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.

    Techniques Used: Staining

    Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).
    Figure Legend Snippet: Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).

    Techniques Used:

    Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).
    Figure Legend Snippet: Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).

    Techniques Used: Produced

    Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.
    Figure Legend Snippet: Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.

    Techniques Used: Staining, Enzyme-linked Immunospot

    Study Design: evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant rhesus macaques. Infant rhesus macaques (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 µg mRNA encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 µg S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of 8 animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18 and 22, and lymph node biopsies were obtained at week 6.
    Figure Legend Snippet: Study Design: evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant rhesus macaques. Infant rhesus macaques (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 µg mRNA encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 µg S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of 8 animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18 and 22, and lymph node biopsies were obtained at week 6.

    Techniques Used:

    Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).
    Figure Legend Snippet: Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).

    Techniques Used: Staining

    S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.
    Figure Legend Snippet: S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.

    Techniques Used:

    Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.
    Figure Legend Snippet: Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.

    Techniques Used: Multiplex Assay, Luminex

    SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).
    Figure Legend Snippet: SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Transformation Assay, Fluorescence

    Vaccine-elicited SARS-CoV-2 neutralization responses. Panel A shows the ID 50 neutralization titers obtained using the pseudovirus neutralization assay. Panel B depicts the ID 50 neutralization titers in infant and adult rhesus macaques vaccinated with the mRNA-LNP vaccine ( 16 ). Infant RM were vaccinated with 30 µg of mRNA-LNP at week 0 and at week 4. Adult RM followed the same vaccine schedule but received 10 µg or 100 µg of the mRNA-LNP vaccine. Neutralization was assessed with a pseudovirus assay at week 8 (4 weeks after the second vaccination). Panel C shows the ID 50 neutralization titers obtained using the whole virus neutralization assay. Panel D illustrates the correlation of ID 50 (left graph) and ID 80 (right graph) neutralizing titers from both vaccine groups obtained in the pseudovirus or whole virus neutralization assay at week 6. Correlation was assessed using Spearman rank test.
    Figure Legend Snippet: Vaccine-elicited SARS-CoV-2 neutralization responses. Panel A shows the ID 50 neutralization titers obtained using the pseudovirus neutralization assay. Panel B depicts the ID 50 neutralization titers in infant and adult rhesus macaques vaccinated with the mRNA-LNP vaccine ( 16 ). Infant RM were vaccinated with 30 µg of mRNA-LNP at week 0 and at week 4. Adult RM followed the same vaccine schedule but received 10 µg or 100 µg of the mRNA-LNP vaccine. Neutralization was assessed with a pseudovirus assay at week 8 (4 weeks after the second vaccination). Panel C shows the ID 50 neutralization titers obtained using the whole virus neutralization assay. Panel D illustrates the correlation of ID 50 (left graph) and ID 80 (right graph) neutralizing titers from both vaccine groups obtained in the pseudovirus or whole virus neutralization assay at week 6. Correlation was assessed using Spearman rank test.

    Techniques Used: Neutralization

    6) Product Images from "High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform"

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    Journal: bioRxiv

    doi: 10.1101/2020.06.16.155580

    Detection of SARS-CoV-2 antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.
    Figure Legend Snippet: Detection of SARS-CoV-2 antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.

    Techniques Used: Fluorescence, Polymerase Chain Reaction, Standard Deviation

    Antibody avidity against SARS-CoV-2 antigens. (a) Avidity of anti-S1 IgG and anti-RBD IgG measured in IgG-positive, PCR-confirmed COVID-19 patient sera collected 6-45 days post symptom onset. The serum of PAMF-065 showed unusually high avidity for anti-S1 IgG while being negative for anti-RBD IgG. (b) Upper panel: Fluorescence images of IgG-only channel showing PAMF-065 serum sample with high anti-S1 IgG level with and without urea treatment, hence high avidity. It showed negligible anti-RBD IgG. Lower panel: Fluorescence images showing another patient serum tested, PAMF-011, with much reduced anti-S1 IgG level after urea treatment, indicating low avidity. Low avidity was observed for all samples except PAMF-065. (c) Anti-S1 IgG median fluorescence intensity (MFI) signals of the PAMF-065 sample with and without urea treatment. The error bars indicate one standard deviation away from the mean.
    Figure Legend Snippet: Antibody avidity against SARS-CoV-2 antigens. (a) Avidity of anti-S1 IgG and anti-RBD IgG measured in IgG-positive, PCR-confirmed COVID-19 patient sera collected 6-45 days post symptom onset. The serum of PAMF-065 showed unusually high avidity for anti-S1 IgG while being negative for anti-RBD IgG. (b) Upper panel: Fluorescence images of IgG-only channel showing PAMF-065 serum sample with high anti-S1 IgG level with and without urea treatment, hence high avidity. It showed negligible anti-RBD IgG. Lower panel: Fluorescence images showing another patient serum tested, PAMF-011, with much reduced anti-S1 IgG level after urea treatment, indicating low avidity. Low avidity was observed for all samples except PAMF-065. (c) Anti-S1 IgG median fluorescence intensity (MFI) signals of the PAMF-065 sample with and without urea treatment. The error bars indicate one standard deviation away from the mean.

    Techniques Used: Polymerase Chain Reaction, Fluorescence, Standard Deviation

    Correlation of antibodies against two SARS-CoV-2 antigens. (a) Correlation plot of anti-S1 IgG level (y-axis) and anti-RBD IgG level (x-axis) measured in PCR-confirmed COVID-19 patient sera. The dashed line was drawn to have a slope of 1. The upper left inset shows the scanned image of the IgG-only channel in a patient serum labeled as PAMF-065, which displayed high signal on the S1 antigen but not on the RBD antigen. The lower right inset shows the scanned image of IgG levels of a sample labeled as PAMF-011, displaying about equal IgG signals against S1 and RBD. (b) Correlation plot of anti-S1 IgM level (y-axis) and anti-RBD IgM level (x-axis) measured in COVID-19 patient sera. The dashed line was drawn to have a slope of 1.
    Figure Legend Snippet: Correlation of antibodies against two SARS-CoV-2 antigens. (a) Correlation plot of anti-S1 IgG level (y-axis) and anti-RBD IgG level (x-axis) measured in PCR-confirmed COVID-19 patient sera. The dashed line was drawn to have a slope of 1. The upper left inset shows the scanned image of the IgG-only channel in a patient serum labeled as PAMF-065, which displayed high signal on the S1 antigen but not on the RBD antigen. The lower right inset shows the scanned image of IgG levels of a sample labeled as PAMF-011, displaying about equal IgG signals against S1 and RBD. (b) Correlation plot of anti-S1 IgM level (y-axis) and anti-RBD IgM level (x-axis) measured in COVID-19 patient sera. The dashed line was drawn to have a slope of 1.

    Techniques Used: Polymerase Chain Reaction, Labeling

    A nano-plasmonic platform for SARS-CoV-2 antibody testing. (a) An overlay of confocal fluorescence scanned images of IgG (green) and IgM (red) channels acquired after testing 16 serum samples in 16 isolated wells (square-shaped regions). Yellowish-green colored spots correspond to the presence of both IgG and IgM in the sample. The lower right schematic drawing shows the printing layout of S1 (in green) and RBD (in blue) antigens and human IgG control spots (in white) in each well. The BSA-biotin spots (in red) are always labeled by a streptavidin dye in the IgM fluorescence channel to serve as an intrawell signal normalizer. (b) Box plots of IgG levels detected in PCR-negative COVID-19 or presumptive negative (‘Healthy’) and PCR-positive (‘PCR+’) COVID-19 samples with the cutoff indicated as a dashed red line. (c) The same as (b) except for IgM. (d) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and 62 PCR-positive COVID-19 serum, which was used to establish IgG and IgM cutoffs. (e) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and PCR-positive COVID-19 serum samples collected 15-45 days post symptom onset.
    Figure Legend Snippet: A nano-plasmonic platform for SARS-CoV-2 antibody testing. (a) An overlay of confocal fluorescence scanned images of IgG (green) and IgM (red) channels acquired after testing 16 serum samples in 16 isolated wells (square-shaped regions). Yellowish-green colored spots correspond to the presence of both IgG and IgM in the sample. The lower right schematic drawing shows the printing layout of S1 (in green) and RBD (in blue) antigens and human IgG control spots (in white) in each well. The BSA-biotin spots (in red) are always labeled by a streptavidin dye in the IgM fluorescence channel to serve as an intrawell signal normalizer. (b) Box plots of IgG levels detected in PCR-negative COVID-19 or presumptive negative (‘Healthy’) and PCR-positive (‘PCR+’) COVID-19 samples with the cutoff indicated as a dashed red line. (c) The same as (b) except for IgM. (d) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and 62 PCR-positive COVID-19 serum, which was used to establish IgG and IgM cutoffs. (e) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and PCR-positive COVID-19 serum samples collected 15-45 days post symptom onset.

    Techniques Used: Fluorescence, Isolation, Labeling, Polymerase Chain Reaction

    Highly sensitive and specific SARS-CoV-2 antibody test. (a) Percentages of samples with IgG/IgM antibody status combinations according to days from symptom onset to sample collection date in a range from 0-7, 8-14, and 15-45 days. (b) Box plots of IgG levels detected in four groups of serum samples indicated on the x-axis with the cutoff displayed as a dashed red line. ‘PCR+’ denotes serum samples from patients who tested positive by PCR for COVID-19 and ‘PCR-’ denotes those who tested negative. ‘Pre-pand.’ corresponds to pre-pandemic collected samples. ‘Cross R.’ corresponds to samples from patients with other diseases for cross-reactivity evaluation. (c) The same as (b) except for IgM.
    Figure Legend Snippet: Highly sensitive and specific SARS-CoV-2 antibody test. (a) Percentages of samples with IgG/IgM antibody status combinations according to days from symptom onset to sample collection date in a range from 0-7, 8-14, and 15-45 days. (b) Box plots of IgG levels detected in four groups of serum samples indicated on the x-axis with the cutoff displayed as a dashed red line. ‘PCR+’ denotes serum samples from patients who tested positive by PCR for COVID-19 and ‘PCR-’ denotes those who tested negative. ‘Pre-pand.’ corresponds to pre-pandemic collected samples. ‘Cross R.’ corresponds to samples from patients with other diseases for cross-reactivity evaluation. (c) The same as (b) except for IgM.

    Techniques Used: Polymerase Chain Reaction

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

    Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.
    Figure Legend Snippet: Graphical illustrations of the COVID-19 related immunoassays that were performed with our microfluidic chemiluminescent ELISA platform, including (A) affinity evaluation of calibration antibodies, (B) detection of circulating anti-SARS-CoV-2 S1 IgG in serum samples, and (C) detection of SARS-CoV-2 antigens such as S1 and N protein.

    Techniques Used: Chemiluminescent ELISA

    8) Product Images from "Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies"

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    Journal: bioRxiv

    doi: 10.1101/2021.04.03.438258

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.
    Figure Legend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Techniques Used: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.
    Figure Legend Snippet: SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Techniques Used: Neutralization, Stable Transfection, Expressing, Infection

    RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).
    Figure Legend Snippet: RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Techniques Used: Incubation, Staining, Imaging, Expressing, Fluorescence, Cryo-EM Sample Prep, Inhibition

    S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).
    Figure Legend Snippet: S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Techniques Used: In Vivo, Injection, Infection, TCID50 Assay

    SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD
    Figure Legend Snippet: SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Techniques Used: Activity Assay, Neutralization, Infection, Incubation, Stable Transfection, Immunostaining, Inhibition, Activation Assay, Reporter Assay

    Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.
    Figure Legend Snippet: Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Techniques Used: Expressing, Infection, In Vitro, Transformation Assay, Transduction, Incubation, Cell Culture, Adsorption, Blocking Assay

    Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.
    Figure Legend Snippet: Characterization of SARS-CoV-2-susceptible cell lines. a , SARS-CoV-2 neutralization with 10 μg/ml of S309, S2E12 and S2X33 on Vero E6 or Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection and viral nucleocapsid protein was immunostained. b , Purified, fluorescently-labelled SARS-CoV-2 spike protein or RBD protein was incubated with the indicated cell lines and protein binding was quantified by flow cytometry. c , Correlation analysis between ACE2 transcript levels and maximum antibody neutralization in all SARS-CoV-2-susceptible cell lines.

    Techniques Used: Neutralization, Infection, Purification, Incubation, Protein Binding, Flow Cytometry

    Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p
    Figure Legend Snippet: Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Techniques Used: Injection, Infection

    HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.
    Figure Legend Snippet: HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Techniques Used: Expressing, Infection, Stable Transfection

    Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.
    Figure Legend Snippet: Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Techniques Used: Binding Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence, Infection, Variant Assay

    DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.
    Figure Legend Snippet: DC-SIGN, L-SIGN and SIGLEC1 function as auxiliary receptors for SARS-CoV-2 infection. a, VSV-SARS-CoV-2 pseudovirus infection of HEK293T cells transfected to over-express ACE2 or a panel of selected lectins and published receptor candidates. b, Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and immunostained at 24 hours for the SARS-CoV-2 nucleocapsid protein (red). c, HEK293T stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified at 24 hours. d, Stable cell lines were incubated with different concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. e, HEK293T, HeLa and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with VSV-SARS-CoV-2 pseudovirus. f, Stable cell lines were treated with ACE2 siRNA followed by infection with VSV-SARS-CoV-2 pseudovirus four days post transfection. g, Stable cell lines were incubated with different concentrations of anti-ACE2 goat polyclonal antibodies and infected with VSV-SARS-CoV-2 pseudovirus.

    Techniques Used: Infection, Transfection, Stable Transfection, Luciferase, Incubation

    Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.
    Figure Legend Snippet: Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Techniques Used:

    9) Product Images from "The Characterization of Disease Severity Associated IgG Subclasses Response in COVID-19 Patients"

    Article Title: The Characterization of Disease Severity Associated IgG Subclasses Response in COVID-19 Patients

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.632814

    IgG1 and IgG3 were the main subclasses induced in COVID-19 patients and related with disease severity. IgG1 (A) and IgG3 (B) responses to NP, S, and RBD and neutralizing antibody (C) against SARS-CoV-2 pseudo-virus with luciferase reporter gene in the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were detected. Differences of medium values between groups were analyzed by Mann-Whitney U -test. Significant correlations among Severity, NAb and IgG subclasses (D) including anti-NP IgG1, IgG3, anti-S IgG1, IgG3, anti-RBD IgG1, IgG3 were shown. Spearman correlation coefficient was calculated. A two-tailed P value
    Figure Legend Snippet: IgG1 and IgG3 were the main subclasses induced in COVID-19 patients and related with disease severity. IgG1 (A) and IgG3 (B) responses to NP, S, and RBD and neutralizing antibody (C) against SARS-CoV-2 pseudo-virus with luciferase reporter gene in the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were detected. Differences of medium values between groups were analyzed by Mann-Whitney U -test. Significant correlations among Severity, NAb and IgG subclasses (D) including anti-NP IgG1, IgG3, anti-S IgG1, IgG3, anti-RBD IgG1, IgG3 were shown. Spearman correlation coefficient was calculated. A two-tailed P value

    Techniques Used: Luciferase, MANN-WHITNEY, Two Tailed Test

    IgA, IgG, and IgM antibodies responses in COVID-19 ranging from asymptomatic to severe patients. Serum samples collected from COVID-19 patients were used for detecting IgA, IgG, and IgM levels to NP (A) , S (B) , and RBD (C) antigens of SARS-CoV-2 via ELISA. Antibody titers of the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were shown in (A–C) . Mann-Whitney U -test was used to compare differences of medium values between groups, a two-tailed P value
    Figure Legend Snippet: IgA, IgG, and IgM antibodies responses in COVID-19 ranging from asymptomatic to severe patients. Serum samples collected from COVID-19 patients were used for detecting IgA, IgG, and IgM levels to NP (A) , S (B) , and RBD (C) antigens of SARS-CoV-2 via ELISA. Antibody titers of the healthy controls ( n = 11) and the severe ( n = 24), moderate ( n = 54), mild ( n = 35), asymptomatic ( n = 10) patients were shown in (A–C) . Mann-Whitney U -test was used to compare differences of medium values between groups, a two-tailed P value

    Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Two Tailed Test

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

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

    Journal: Signal Transduction and Targeted Therapy

    doi: 10.1038/s41392-020-00301-9

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

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

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

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

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

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

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    Recombinant:

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    Concentration Assay:

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    Blocking Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
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    Microarray:

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    Binding Assay:

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    Enzyme-linked Immunosorbent Assay:

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    Injection:

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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    Detection of <t>SARS-CoV-2</t> antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.
    Sars Cov 2 2019 Ncov Spike Rbd His Recombinant Protein Covid 19 Spike Rbd Research, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 spike
    Genome-wide CRISPR/Cas9 screen identifies host factors using Sdel virus as model. a Schematic of the screening process. A549 cells expressing the human ACE2 were used to generate the CRISPR sgRNA knockout cell library. The library was infected with Sdel strain of <t>SARS-CoV-2,</t> and cells survived were harvested for genomic extraction and sequence analysis. b Genes and complexes identified from the CRISPR screen. The top 32 (FDR
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    Detection of SARS-CoV-2 antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.

    Journal: bioRxiv

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    doi: 10.1101/2020.06.16.155580

    Figure Lengend Snippet: Detection of SARS-CoV-2 antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.

    Article Snippet: Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.).

    Techniques: Fluorescence, Polymerase Chain Reaction, Standard Deviation

    Antibody avidity against SARS-CoV-2 antigens. (a) Avidity of anti-S1 IgG and anti-RBD IgG measured in IgG-positive, PCR-confirmed COVID-19 patient sera collected 6-45 days post symptom onset. The serum of PAMF-065 showed unusually high avidity for anti-S1 IgG while being negative for anti-RBD IgG. (b) Upper panel: Fluorescence images of IgG-only channel showing PAMF-065 serum sample with high anti-S1 IgG level with and without urea treatment, hence high avidity. It showed negligible anti-RBD IgG. Lower panel: Fluorescence images showing another patient serum tested, PAMF-011, with much reduced anti-S1 IgG level after urea treatment, indicating low avidity. Low avidity was observed for all samples except PAMF-065. (c) Anti-S1 IgG median fluorescence intensity (MFI) signals of the PAMF-065 sample with and without urea treatment. The error bars indicate one standard deviation away from the mean.

    Journal: bioRxiv

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    doi: 10.1101/2020.06.16.155580

    Figure Lengend Snippet: Antibody avidity against SARS-CoV-2 antigens. (a) Avidity of anti-S1 IgG and anti-RBD IgG measured in IgG-positive, PCR-confirmed COVID-19 patient sera collected 6-45 days post symptom onset. The serum of PAMF-065 showed unusually high avidity for anti-S1 IgG while being negative for anti-RBD IgG. (b) Upper panel: Fluorescence images of IgG-only channel showing PAMF-065 serum sample with high anti-S1 IgG level with and without urea treatment, hence high avidity. It showed negligible anti-RBD IgG. Lower panel: Fluorescence images showing another patient serum tested, PAMF-011, with much reduced anti-S1 IgG level after urea treatment, indicating low avidity. Low avidity was observed for all samples except PAMF-065. (c) Anti-S1 IgG median fluorescence intensity (MFI) signals of the PAMF-065 sample with and without urea treatment. The error bars indicate one standard deviation away from the mean.

    Article Snippet: Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.).

    Techniques: Polymerase Chain Reaction, Fluorescence, Standard Deviation

    Correlation of antibodies against two SARS-CoV-2 antigens. (a) Correlation plot of anti-S1 IgG level (y-axis) and anti-RBD IgG level (x-axis) measured in PCR-confirmed COVID-19 patient sera. The dashed line was drawn to have a slope of 1. The upper left inset shows the scanned image of the IgG-only channel in a patient serum labeled as PAMF-065, which displayed high signal on the S1 antigen but not on the RBD antigen. The lower right inset shows the scanned image of IgG levels of a sample labeled as PAMF-011, displaying about equal IgG signals against S1 and RBD. (b) Correlation plot of anti-S1 IgM level (y-axis) and anti-RBD IgM level (x-axis) measured in COVID-19 patient sera. The dashed line was drawn to have a slope of 1.

    Journal: bioRxiv

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    doi: 10.1101/2020.06.16.155580

    Figure Lengend Snippet: Correlation of antibodies against two SARS-CoV-2 antigens. (a) Correlation plot of anti-S1 IgG level (y-axis) and anti-RBD IgG level (x-axis) measured in PCR-confirmed COVID-19 patient sera. The dashed line was drawn to have a slope of 1. The upper left inset shows the scanned image of the IgG-only channel in a patient serum labeled as PAMF-065, which displayed high signal on the S1 antigen but not on the RBD antigen. The lower right inset shows the scanned image of IgG levels of a sample labeled as PAMF-011, displaying about equal IgG signals against S1 and RBD. (b) Correlation plot of anti-S1 IgM level (y-axis) and anti-RBD IgM level (x-axis) measured in COVID-19 patient sera. The dashed line was drawn to have a slope of 1.

    Article Snippet: Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.).

    Techniques: Polymerase Chain Reaction, Labeling

    A nano-plasmonic platform for SARS-CoV-2 antibody testing. (a) An overlay of confocal fluorescence scanned images of IgG (green) and IgM (red) channels acquired after testing 16 serum samples in 16 isolated wells (square-shaped regions). Yellowish-green colored spots correspond to the presence of both IgG and IgM in the sample. The lower right schematic drawing shows the printing layout of S1 (in green) and RBD (in blue) antigens and human IgG control spots (in white) in each well. The BSA-biotin spots (in red) are always labeled by a streptavidin dye in the IgM fluorescence channel to serve as an intrawell signal normalizer. (b) Box plots of IgG levels detected in PCR-negative COVID-19 or presumptive negative (‘Healthy’) and PCR-positive (‘PCR+’) COVID-19 samples with the cutoff indicated as a dashed red line. (c) The same as (b) except for IgM. (d) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and 62 PCR-positive COVID-19 serum, which was used to establish IgG and IgM cutoffs. (e) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and PCR-positive COVID-19 serum samples collected 15-45 days post symptom onset.

    Journal: bioRxiv

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    doi: 10.1101/2020.06.16.155580

    Figure Lengend Snippet: A nano-plasmonic platform for SARS-CoV-2 antibody testing. (a) An overlay of confocal fluorescence scanned images of IgG (green) and IgM (red) channels acquired after testing 16 serum samples in 16 isolated wells (square-shaped regions). Yellowish-green colored spots correspond to the presence of both IgG and IgM in the sample. The lower right schematic drawing shows the printing layout of S1 (in green) and RBD (in blue) antigens and human IgG control spots (in white) in each well. The BSA-biotin spots (in red) are always labeled by a streptavidin dye in the IgM fluorescence channel to serve as an intrawell signal normalizer. (b) Box plots of IgG levels detected in PCR-negative COVID-19 or presumptive negative (‘Healthy’) and PCR-positive (‘PCR+’) COVID-19 samples with the cutoff indicated as a dashed red line. (c) The same as (b) except for IgM. (d) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and 62 PCR-positive COVID-19 serum, which was used to establish IgG and IgM cutoffs. (e) ROC curve for pGOLD SARS-CoV-2 IgG/IgM assay based on 384 negative and PCR-positive COVID-19 serum samples collected 15-45 days post symptom onset.

    Article Snippet: Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.).

    Techniques: Fluorescence, Isolation, Labeling, Polymerase Chain Reaction

    Highly sensitive and specific SARS-CoV-2 antibody test. (a) Percentages of samples with IgG/IgM antibody status combinations according to days from symptom onset to sample collection date in a range from 0-7, 8-14, and 15-45 days. (b) Box plots of IgG levels detected in four groups of serum samples indicated on the x-axis with the cutoff displayed as a dashed red line. ‘PCR+’ denotes serum samples from patients who tested positive by PCR for COVID-19 and ‘PCR-’ denotes those who tested negative. ‘Pre-pand.’ corresponds to pre-pandemic collected samples. ‘Cross R.’ corresponds to samples from patients with other diseases for cross-reactivity evaluation. (c) The same as (b) except for IgM.

    Journal: bioRxiv

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform

    doi: 10.1101/2020.06.16.155580

    Figure Lengend Snippet: Highly sensitive and specific SARS-CoV-2 antibody test. (a) Percentages of samples with IgG/IgM antibody status combinations according to days from symptom onset to sample collection date in a range from 0-7, 8-14, and 15-45 days. (b) Box plots of IgG levels detected in four groups of serum samples indicated on the x-axis with the cutoff displayed as a dashed red line. ‘PCR+’ denotes serum samples from patients who tested positive by PCR for COVID-19 and ‘PCR-’ denotes those who tested negative. ‘Pre-pand.’ corresponds to pre-pandemic collected samples. ‘Cross R.’ corresponds to samples from patients with other diseases for cross-reactivity evaluation. (c) The same as (b) except for IgM.

    Article Snippet: Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.).

    Techniques: Polymerase Chain Reaction

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-021-21213-4

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-021-21213-4

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

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

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

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-021-21213-4

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

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

    Techniques: Infection

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-021-21213-4

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

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

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