sars cov s1  (Sino Biological)


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    Name:
    Human SARS Coronavirus Spike S1 Subunit Insect Cell Lysate
    Description:
    Baculovirus Insect Cell lysate that Human SARS Coronavirus Spike S1 Subunit transfected overexpressed for Western blot WB positive control The whole cell lysate is provided in 1X Sample Buffer 1X modified RIPA buffer 1X SDS loading buffer
    Catalog Number:
    40150-v08b1l
    Product Aliases:
    SARS coronavirus s1 Overexpression Lysate, SARS coronavirus s2 Overexpression Lysate, SARS coronavirus spike Overexpression Lysate, SARS cov spike Overexpression Lysate, SARS ncov RBD Overexpression Lysate, SARS ncov s1 Overexpression Lysate, SARS ncov s2 Overexpression Lysate, SARS ncov spike Overexpression Lysate, SARS novel coronavirus RBD Overexpression Lysate, SARS novel coronavirus s1 Overexpression Lysate, SARS novel coronavirus s2 Overexpression Lysate, SARS novel coronavirus spike Overexpression Lysate, SARS RBD Overexpression Lysate, SARS S1 Overexpression Lysate, SARS s2 Overexpression Lysate, SARS Spike RBD Overexpression Lysate
    Price:
    195
    Applications:
    WB
    Size:
    300µg
    Category:
    Lysate
    Cell Type:
    Baculovirus-Insect cells
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    Structured Review

    Sino Biological sars cov s1
    S1 protein detection. (A) Illustration of the assay mechanism. The sample-to-answer time of this assay is 20 minutes. (B) Entire dynamic ranges of <t>SARS-CoV-2</t> S1 protein (red squares) and SARS-CoV S1 protein (black circles) in 10 times diluted human serum. The averaged background is subtracted from all data points. The solid lines are the linear fit of the data in the log-log scale. The grey shaded area marks 3×standard deviation of the background. The lower limit of detection (LLOD) for SARS-CoV-2 S1 protein and SARS-CoV S1 is 0.4 ng/mL and 0.2 ng/mL, respectively. (C) Calibration curves for S1 proteins between 0.78 and 200 ng/mL. The error bars are generated from duplicate measurements.
    Baculovirus Insect Cell lysate that Human SARS Coronavirus Spike S1 Subunit transfected overexpressed for Western blot WB positive control The whole cell lysate is provided in 1X Sample Buffer 1X modified RIPA buffer 1X SDS loading buffer
    https://www.bioz.com/result/sars cov s1/product/Sino Biological
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    sars cov s1 - by Bioz Stars, 2021-02
    93/100 stars

    Images

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

    3) Product Images from "Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2"

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.12.30.424862

    Screening SARS-CoV-2 antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p
    Figure Legend Snippet: Screening SARS-CoV-2 antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p

    Techniques Used: Activity Assay, Infection, Incubation, CTG Assay, Inhibition

    (A) HPMEC, (B) BEAS-2B, (C) HCT-116, (D) LNCaP, (E) HaCaT, (F) RD, (G) NCI-H1437, (H) Huh-7.5.1, (I) Caco-2, (J) A549/hACE2, (K) HBEC-30KT, or (L) A549 cells were infected with SARS-CoV-2 at MOI 0.5 or 0.05 as in figure 1 . Viral titers were analyzed by TCID50 assay at the indicated time points (hours post-infection, hpi). Dashed lines represent limit of detection. Data represent mean ± SEM for n = 2 independent experiments.
    Figure Legend Snippet: (A) HPMEC, (B) BEAS-2B, (C) HCT-116, (D) LNCaP, (E) HaCaT, (F) RD, (G) NCI-H1437, (H) Huh-7.5.1, (I) Caco-2, (J) A549/hACE2, (K) HBEC-30KT, or (L) A549 cells were infected with SARS-CoV-2 at MOI 0.5 or 0.05 as in figure 1 . Viral titers were analyzed by TCID50 assay at the indicated time points (hours post-infection, hpi). Dashed lines represent limit of detection. Data represent mean ± SEM for n = 2 independent experiments.

    Techniques Used: Infection, TCID50 Assay

    Dose response curves of compounds with SARS-CoV-2 antiviral activity. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: Dose response curves of compounds with SARS-CoV-2 antiviral activity. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Activity Assay, Infection, Inhibition

    (A) Clinical status of compounds tested. (B-C) Remdesivir dose response curves in (B) Vero-E6 and (C) Calu-3 cells showing % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: (A) Clinical status of compounds tested. (B-C) Remdesivir dose response curves in (B) Vero-E6 and (C) Calu-3 cells showing % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Inhibition, Infection

    Confirmation and characterization of SARS-CoV-2 antiviral candidate compounds. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05, treated with the top 12 compounds (shown in Figure 3 ), disulfiram, or apilimod mesylate at indicated concentrations and supernatants were collected at 24 hpi. Viral titers and genome copies were calculated by TCID50 and qRT-PCR, respectively. (A) and (B) protein kinase and protease inhibitors, (C) and (D) anti-inflammatory compounds, (E) and (F) direct-acting antivirals and (G) and (H) other host-targeting compounds. TCID50 data represent mean ± SD for n = 2 independent experiments. Genome copy data represent mean ± SEM for n = 2 technical replicates and are representative of n = 2 independent experiments.
    Figure Legend Snippet: Confirmation and characterization of SARS-CoV-2 antiviral candidate compounds. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05, treated with the top 12 compounds (shown in Figure 3 ), disulfiram, or apilimod mesylate at indicated concentrations and supernatants were collected at 24 hpi. Viral titers and genome copies were calculated by TCID50 and qRT-PCR, respectively. (A) and (B) protein kinase and protease inhibitors, (C) and (D) anti-inflammatory compounds, (E) and (F) direct-acting antivirals and (G) and (H) other host-targeting compounds. TCID50 data represent mean ± SD for n = 2 independent experiments. Genome copy data represent mean ± SEM for n = 2 technical replicates and are representative of n = 2 independent experiments.

    Techniques Used: Infection, Quantitative RT-PCR

    Permissive cell lines to SARS-CoV2 infection. (A) Vero-E6, (B) Calu-3, (C) Huh-7 and (D) HPMEC/hACE2 cells were seeded in 24-well plates and infected with SARS-CoV-2 at MOI 0.5 or 0.05 at 37°C and 5% C0 2 for 30 minutes. Viral inoculum was then removed, cells were washed once in 1x PBS, and 1 ml of regular media was replaced. At the indicated time points (hours post-infection, hpi), plates were freeze/thawed and viral titers from whole cell lysates were analyzed by TCID50 assay. Dashed line represents limit of detection of the assay. Data represent mean ± SEM for n = 2 independent experiments.
    Figure Legend Snippet: Permissive cell lines to SARS-CoV2 infection. (A) Vero-E6, (B) Calu-3, (C) Huh-7 and (D) HPMEC/hACE2 cells were seeded in 24-well plates and infected with SARS-CoV-2 at MOI 0.5 or 0.05 at 37°C and 5% C0 2 for 30 minutes. Viral inoculum was then removed, cells were washed once in 1x PBS, and 1 ml of regular media was replaced. At the indicated time points (hours post-infection, hpi), plates were freeze/thawed and viral titers from whole cell lysates were analyzed by TCID50 assay. Dashed line represents limit of detection of the assay. Data represent mean ± SEM for n = 2 independent experiments.

    Techniques Used: Infection, TCID50 Assay

    Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Infection, Inhibition

    Cell-type specificity of compounds antiviral activity. Huh7, HPMEC/hACE2 and Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with (A) Dinaciclib, (B) BFH772, (C) Budesonide, (D) GC376 sodium, (E) Apilimod mesylate, (F) GKT137831, (G) Cyclosporin A, (H) B02, and (I) Camostat mesylate at indicated concentrations. At 48hpi cells were washed, fixed, and stained with DAPI and for SARS-CoV-2 nucleocapsid protein. Plates were fluorescently imaged and analyzed for nucleocapsid stain per nuclei. Relative infection (full lines) and relative number of cells (dashed lines) are normalized to DMSO-treated wells. Data represent mean ± SEM for n = 4 technical replicates and are representative of n = 3 independent experiments.
    Figure Legend Snippet: Cell-type specificity of compounds antiviral activity. Huh7, HPMEC/hACE2 and Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with (A) Dinaciclib, (B) BFH772, (C) Budesonide, (D) GC376 sodium, (E) Apilimod mesylate, (F) GKT137831, (G) Cyclosporin A, (H) B02, and (I) Camostat mesylate at indicated concentrations. At 48hpi cells were washed, fixed, and stained with DAPI and for SARS-CoV-2 nucleocapsid protein. Plates were fluorescently imaged and analyzed for nucleocapsid stain per nuclei. Relative infection (full lines) and relative number of cells (dashed lines) are normalized to DMSO-treated wells. Data represent mean ± SEM for n = 4 technical replicates and are representative of n = 3 independent experiments.

    Techniques Used: Activity Assay, Infection, Staining

    B02 synergy with remdesivir. (A) Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with 2 μM remdesivir, 10 μM B02, or a combination of 2 μM remdesivir and 10 μM B02 for 72h. CPE inhibition was measured by CTG assay. (B) Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with remdesivir at indicated concentrations in the presence or absence of 10 μM B02 for 96h. CPE inhibition was measured by CTG assay and was normalized to DMSO-treated wells. Data represent mean ± SD for n = 2 technical replicates.
    Figure Legend Snippet: B02 synergy with remdesivir. (A) Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with 2 μM remdesivir, 10 μM B02, or a combination of 2 μM remdesivir and 10 μM B02 for 72h. CPE inhibition was measured by CTG assay. (B) Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with remdesivir at indicated concentrations in the presence or absence of 10 μM B02 for 96h. CPE inhibition was measured by CTG assay and was normalized to DMSO-treated wells. Data represent mean ± SD for n = 2 technical replicates.

    Techniques Used: Infection, Inhibition, CTG Assay

    4) Product Images from "Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without"

    Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.340604

    Spike mediated particle entry ( A ) Generation of pseudotyped lentiviral vectors. Second generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor binding domain (RBD, orange) are indicated. ( B ) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s, bottom blot for 5 s. ( C ) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μl vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Grey shaded area indicates 95% CI of signals from untransduced cells (blanks). ( D ) Effect of ACE2-overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95% CIs.
    Figure Legend Snippet: Spike mediated particle entry ( A ) Generation of pseudotyped lentiviral vectors. Second generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor binding domain (RBD, orange) are indicated. ( B ) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s, bottom blot for 5 s. ( C ) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μl vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Grey shaded area indicates 95% CI of signals from untransduced cells (blanks). ( D ) Effect of ACE2-overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95% CIs.

    Techniques Used: Generated, Transfection, Plasmid Preparation, Binding Assay, Western Blot, Staining, Activity Assay, Over Expression, Expressing, Incubation

    5) Product Images from "Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19"

    Article Title: Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19545-8

    Family of symptomatic SARS-CoV-2 PCR positive parents and SARS-CoV-2 PCR negative children have distinct serological responses compared to healthy individuals, characterized by elevated SARS-CoV-2 specific responses. a PLSDA scores plot of healthy (blue triangles) vs family (circles) containing both SARS-CoV-2 PCR positive parents (orange) and negative children (yellow) exhibited 98.0% calibration and 96.0% cross-validation accuracy, with 62.7% of variance explained by LV1 (x-axis). Family member samples are labeled with A (adult) or C (child) with the day of sample collection listed after D. b PLSDA plot of LV1 loadings driving the separation of groups, where negatively loaded features are associated with the family members. c Hierarchical clustering of healthy individuals (blue) and family members (parents, orange; children, yellow) using a feature-selected serological signature, where red indicates a relatively high antibody response and blue a relatively low antibody response (z-score). Samples (x-axis) are labeled with H (healthy non-household members), and A (adult) or C (child). Day of sample collection is listed at the end of family member sample labels.
    Figure Legend Snippet: Family of symptomatic SARS-CoV-2 PCR positive parents and SARS-CoV-2 PCR negative children have distinct serological responses compared to healthy individuals, characterized by elevated SARS-CoV-2 specific responses. a PLSDA scores plot of healthy (blue triangles) vs family (circles) containing both SARS-CoV-2 PCR positive parents (orange) and negative children (yellow) exhibited 98.0% calibration and 96.0% cross-validation accuracy, with 62.7% of variance explained by LV1 (x-axis). Family member samples are labeled with A (adult) or C (child) with the day of sample collection listed after D. b PLSDA plot of LV1 loadings driving the separation of groups, where negatively loaded features are associated with the family members. c Hierarchical clustering of healthy individuals (blue) and family members (parents, orange; children, yellow) using a feature-selected serological signature, where red indicates a relatively high antibody response and blue a relatively low antibody response (z-score). Samples (x-axis) are labeled with H (healthy non-household members), and A (adult) or C (child). Day of sample collection is listed at the end of family member sample labels.

    Techniques Used: Polymerase Chain Reaction, Labeling

    Salivary and plasma antibody responses against SARS-CoV-2 S1 protein by ELISA and by microneutralization assay. a Anti-S1 salivary IgA, IgG, and IgM. # IgA anti-S1 response that developed concurrent with resolution of symptoms. b Anti-S1 plasma IgA, IgG, and IgM. c Neutralizing antibody activity in plasma. A1: mother, A2: father, C1: male (9 years), C2: male (7 years), C3: female (5 years), (P) positive control.
    Figure Legend Snippet: Salivary and plasma antibody responses against SARS-CoV-2 S1 protein by ELISA and by microneutralization assay. a Anti-S1 salivary IgA, IgG, and IgM. # IgA anti-S1 response that developed concurrent with resolution of symptoms. b Anti-S1 plasma IgA, IgG, and IgM. c Neutralizing antibody activity in plasma. A1: mother, A2: father, C1: male (9 years), C2: male (7 years), C3: female (5 years), (P) positive control.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Activity Assay, Positive Control

    6) Product Images from "Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals"

    Article Title: Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

    Journal: Vaccines

    doi: 10.3390/vaccines8040684

    Antibody responses in patients with COVID-19. ( A ) Levels of serum IgG, IgA, and IgM antibodies against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 were measured by ELISA. Blue (IgG), green (IgA), and purple (IgM) lines represent the antibody titers of individual COVID-19 positive sera, pink lines represent negative control sera. ( B ) Each dot represents an individual antibody titer, and groups are divided by age and sex. ( C ) Correlation of anti-N responses with other antiviral protein responses in patients with COVID-19. Correlations of the values were assessed by linear regression. ( D ) Correlations between neutralization titer (x-axis) and each antibody response to the viral protein of SARS-CoV-2 (y-axis) are shown. Each dot indicates the neutralization titer of serum antibodies of patients with COVID-19, as determined by calculating the highest dilution of serum that prevents infection of 50% of inoculations and antiviral protein titers (IgG) of human sera, as determined by ELISA.
    Figure Legend Snippet: Antibody responses in patients with COVID-19. ( A ) Levels of serum IgG, IgA, and IgM antibodies against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 were measured by ELISA. Blue (IgG), green (IgA), and purple (IgM) lines represent the antibody titers of individual COVID-19 positive sera, pink lines represent negative control sera. ( B ) Each dot represents an individual antibody titer, and groups are divided by age and sex. ( C ) Correlation of anti-N responses with other antiviral protein responses in patients with COVID-19. Correlations of the values were assessed by linear regression. ( D ) Correlations between neutralization titer (x-axis) and each antibody response to the viral protein of SARS-CoV-2 (y-axis) are shown. Each dot indicates the neutralization titer of serum antibodies of patients with COVID-19, as determined by calculating the highest dilution of serum that prevents infection of 50% of inoculations and antiviral protein titers (IgG) of human sera, as determined by ELISA.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization, Infection

    Immune responses of mice to SARS-CoV-2. ( A ) Viral protein-specific IgG antibody responses of mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2. Viral protein-specific IgG responses of murine sera and cross-reactivities to the viral proteins of SARS-CoV, CCoV 1-71, HCoV NL63, and influenza virus were measured by ELISA. Blue bars represent homologous antibody responses, and bars with slash lines display cross-reactive antibody responses. Red lines are the cut-offs calculated as 3 times the mean absorbance value of the preimmune sera. ( B ) Antibody response was measured in mice immunized with S, N, E, M, or 3CLpro of SARS-CoV. Purple bars show homologous antibody responses, and bars with slash lines represent cross-reactive antibody responses. ( C ) S-, S1-, RBD-, S2-, N-, E-, and NS3-specific IgG PCs in spinal bone marrow (left) and ASCs in cervical lymph nodes (right) were quantified ex vivo in ELISpot assays. Bone marrow and lymph node cells were obtained from the mice immunized with respective viral protein of SARS-CoV-2. ( D ) The presence of neutralizing antibodies in mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2; inactivated whole virus of SARS-CoV-2; and S, N, E, M, or 3CLpro of SARS-CoV was measured by in vitro neutralization assays using a pseudotyped virus. The transduction efficacy was determined by measuring the luciferase activity. Nonlinear regression analysis was used to obtain the IC 50 in the neutralization assays.
    Figure Legend Snippet: Immune responses of mice to SARS-CoV-2. ( A ) Viral protein-specific IgG antibody responses of mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2. Viral protein-specific IgG responses of murine sera and cross-reactivities to the viral proteins of SARS-CoV, CCoV 1-71, HCoV NL63, and influenza virus were measured by ELISA. Blue bars represent homologous antibody responses, and bars with slash lines display cross-reactive antibody responses. Red lines are the cut-offs calculated as 3 times the mean absorbance value of the preimmune sera. ( B ) Antibody response was measured in mice immunized with S, N, E, M, or 3CLpro of SARS-CoV. Purple bars show homologous antibody responses, and bars with slash lines represent cross-reactive antibody responses. ( C ) S-, S1-, RBD-, S2-, N-, E-, and NS3-specific IgG PCs in spinal bone marrow (left) and ASCs in cervical lymph nodes (right) were quantified ex vivo in ELISpot assays. Bone marrow and lymph node cells were obtained from the mice immunized with respective viral protein of SARS-CoV-2. ( D ) The presence of neutralizing antibodies in mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2; inactivated whole virus of SARS-CoV-2; and S, N, E, M, or 3CLpro of SARS-CoV was measured by in vitro neutralization assays using a pseudotyped virus. The transduction efficacy was determined by measuring the luciferase activity. Nonlinear regression analysis was used to obtain the IC 50 in the neutralization assays.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Enzyme-linked Immunospot, In Vitro, Neutralization, Transduction, Luciferase, Activity Assay

    Antibody responses in dogs and cats. ( A ) Serum antibody responses of dogs to S, S1, RBD, S2, N, E, and NS3 viral proteins of SARS-CoV-2, and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by IgG ELISA. The OD values were normalized by subtracting the OD values generated by sera from SPF dogs. ( B ) In canine serum samples, IgG levels against CCoV/HCoV and those against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2, as detected by ELISA, were used in correlation analyses. ( C ) Serum IgG antibody responses of cats to SARS-CoV-2 S, S1, RBD, S2, N, E, and NS3, and and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by ELISA. The crude OD values generated by ELISA were normalized by subtracting the OD values generated by sera from SPF cats. ( D ) ELISA was also used to determine IgG antibody levels against N of SARS-CoV-2, CCoV 1-71, and HCoV NL63 in the sera of cats for correlation analyses.
    Figure Legend Snippet: Antibody responses in dogs and cats. ( A ) Serum antibody responses of dogs to S, S1, RBD, S2, N, E, and NS3 viral proteins of SARS-CoV-2, and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by IgG ELISA. The OD values were normalized by subtracting the OD values generated by sera from SPF dogs. ( B ) In canine serum samples, IgG levels against CCoV/HCoV and those against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2, as detected by ELISA, were used in correlation analyses. ( C ) Serum IgG antibody responses of cats to SARS-CoV-2 S, S1, RBD, S2, N, E, and NS3, and and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by ELISA. The crude OD values generated by ELISA were normalized by subtracting the OD values generated by sera from SPF cats. ( D ) ELISA was also used to determine IgG antibody levels against N of SARS-CoV-2, CCoV 1-71, and HCoV NL63 in the sera of cats for correlation analyses.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Generated

    Preexisting and cross-reactive antibodies to human respiratory viruses in sera of patients with COVID-19. ( A ) Respiratory virus-specific antibody profiling. Heatmap indicating IgG antibody responses in 46 sera samples (37 COVID-19 samples and 9 control samples) measured by protein microarray. The microarray slides were probed with human sera and labeled with secondary antibodies to human IgG conjugated to a quantum dot fluorophore. The slides were imaged using the GenePix 4000B Microarray Scanner to measure background-subtracted median spot fluorescence. Mean fluorescence intensity (MFI) of the 4 replicates for each antigen was used for analysis. Each column represents a paired sample, and each row, a respiratory virus protein. Box graph indicates the sum of antibody responses of all sera samples for each respiratory virus protein. ( B ) Correlation between protein microarray and ELISA-based readouts. Each dot indicates the antibody response of serum from a single patient with COVID-19 against S, S1, RBD, S2, and N of SARS-CoV-2, as measured by fluorescence intensity (y-axis) and absorbance at 450 nm, as determined by ELISA (x-axis). ( C ) Relations of antibody response between specific antibody levels against SARS-CoV-2 and those against endemic HCoVs NL63, 229E, HKU1, and OC43 in patients with COVID-19.
    Figure Legend Snippet: Preexisting and cross-reactive antibodies to human respiratory viruses in sera of patients with COVID-19. ( A ) Respiratory virus-specific antibody profiling. Heatmap indicating IgG antibody responses in 46 sera samples (37 COVID-19 samples and 9 control samples) measured by protein microarray. The microarray slides were probed with human sera and labeled with secondary antibodies to human IgG conjugated to a quantum dot fluorophore. The slides were imaged using the GenePix 4000B Microarray Scanner to measure background-subtracted median spot fluorescence. Mean fluorescence intensity (MFI) of the 4 replicates for each antigen was used for analysis. Each column represents a paired sample, and each row, a respiratory virus protein. Box graph indicates the sum of antibody responses of all sera samples for each respiratory virus protein. ( B ) Correlation between protein microarray and ELISA-based readouts. Each dot indicates the antibody response of serum from a single patient with COVID-19 against S, S1, RBD, S2, and N of SARS-CoV-2, as measured by fluorescence intensity (y-axis) and absorbance at 450 nm, as determined by ELISA (x-axis). ( C ) Relations of antibody response between specific antibody levels against SARS-CoV-2 and those against endemic HCoVs NL63, 229E, HKU1, and OC43 in patients with COVID-19.

    Techniques Used: Microarray, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay

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

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.599587

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

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

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

    Techniques Used: Mouse Assay, Incubation, Activity Assay

    8) Product Images from "SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques"

    Article Title: SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20642-x

    IgG1 subclass and neutralizing antibodies induced following SARS-CoV-2 infection. A Fold increase in antibody responses in animals was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Data shown are n = 8 animals for all time points. Horizontal line indicates median B Fold increase in IgG1, IgG2, IgG3, and IgG4 antibodies against S1, S2, and N show dominance of IgG1 subclass antibodies. Data shown are for n = 6 animals not given CP. C Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-receptor binding domain (RBD) IgG antibodies measured by ELISA. Unique symbols identify animals in each of the experimental groups (two-tailed Pearson test p values shown; correlation for anti-RBD IgG and T h 1 T fh cells shows one-tailed Spearman test p value).
    Figure Legend Snippet: IgG1 subclass and neutralizing antibodies induced following SARS-CoV-2 infection. A Fold increase in antibody responses in animals was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Data shown are n = 8 animals for all time points. Horizontal line indicates median B Fold increase in IgG1, IgG2, IgG3, and IgG4 antibodies against S1, S2, and N show dominance of IgG1 subclass antibodies. Data shown are for n = 6 animals not given CP. C Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-receptor binding domain (RBD) IgG antibodies measured by ELISA. Unique symbols identify animals in each of the experimental groups (two-tailed Pearson test p values shown; correlation for anti-RBD IgG and T h 1 T fh cells shows one-tailed Spearman test p value).

    Techniques Used: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, One-tailed Test

    Humoral responses to SARS-CoV-2 are dominated by IgG antibodies. Concentrations of A IgM, B IgG, and C IgA antibodies (Ab) specific for S1, S2, and N proteins measured by BAMA or ELISA in serum. The dashed line represents the median pre-infection (day 0) concentration for all animals. Unique symbols identify animals in each of the experimental groups. (** p = 0.007, * p = 0.015 at indicated time points relative to d0 using a Wilcoxon matched-pairs signed-rank two-tailed t test).
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 are dominated by IgG antibodies. Concentrations of A IgM, B IgG, and C IgA antibodies (Ab) specific for S1, S2, and N proteins measured by BAMA or ELISA in serum. The dashed line represents the median pre-infection (day 0) concentration for all animals. Unique symbols identify animals in each of the experimental groups. (** p = 0.007, * p = 0.015 at indicated time points relative to d0 using a Wilcoxon matched-pairs signed-rank two-tailed t test).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Concentration Assay, Two Tailed Test

    CD4 T fh cells targeting spike (S) and nucleocapsid (N) in blood following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with spike (S) and nucleocapsid (N) peptide pools in PBMCs. B AIM + CXCR5 − and CXCR5 + CD4 subsets in PBMCs at Day 7.
    Figure Legend Snippet: CD4 T fh cells targeting spike (S) and nucleocapsid (N) in blood following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with spike (S) and nucleocapsid (N) peptide pools in PBMCs. B AIM + CXCR5 − and CXCR5 + CD4 subsets in PBMCs at Day 7.

    Techniques Used: Infection

    SARS-CoV-2 infection induces germinal center responses targeting spike (S) and nucleocapsid (N) in mediastinal lymph nodes. A Median fluorescence intensity (MFI) of CXCR5, CCR7, CD69 and B SLAM, ICOS, CD28 within CXCR3 - (orange) and CXCR3 + (magenta) GC T fh cells in spleen following SARS-CoV-2 infection. Naive CD4 T cells in spleen shown for comparison (grey) (SLAM; **** p
    Figure Legend Snippet: SARS-CoV-2 infection induces germinal center responses targeting spike (S) and nucleocapsid (N) in mediastinal lymph nodes. A Median fluorescence intensity (MFI) of CXCR5, CCR7, CD69 and B SLAM, ICOS, CD28 within CXCR3 - (orange) and CXCR3 + (magenta) GC T fh cells in spleen following SARS-CoV-2 infection. Naive CD4 T cells in spleen shown for comparison (grey) (SLAM; **** p

    Techniques Used: Infection, Fluorescence

    Induction of T h 1 CD4 effectors in the lungs during SARS-CoV-2 infection. A Gating strategy for identifying CD95 + CD69 + CD4 and CD8 cells expressing granzyme B, PD-1, α4β7, CCR6, and CXCR3. Fluorochromes used were CD45-A488, CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CD69-BV711, Granzyme B- BV421, PD-1-Pe Cy7, a4b7-PE, CD25-APC, CCR6-PECF594, CXCR3-BV786. B Percentage of CD4 and CD8 T cells expressing granzyme B, PD-1, CXCR3, and CCR6 in lung and blood (* p = 0.02 using a two-tailed Mann–Whitney U test). C Correlation plot of vRNA from nasal washes and either granzyme B (GzmB) or PD-1 in CD8 T cells (one-tailed Pearson test p values shown).
    Figure Legend Snippet: Induction of T h 1 CD4 effectors in the lungs during SARS-CoV-2 infection. A Gating strategy for identifying CD95 + CD69 + CD4 and CD8 cells expressing granzyme B, PD-1, α4β7, CCR6, and CXCR3. Fluorochromes used were CD45-A488, CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CD69-BV711, Granzyme B- BV421, PD-1-Pe Cy7, a4b7-PE, CD25-APC, CCR6-PECF594, CXCR3-BV786. B Percentage of CD4 and CD8 T cells expressing granzyme B, PD-1, CXCR3, and CCR6 in lung and blood (* p = 0.02 using a two-tailed Mann–Whitney U test). C Correlation plot of vRNA from nasal washes and either granzyme B (GzmB) or PD-1 in CD8 T cells (one-tailed Pearson test p values shown).

    Techniques Used: Infection, Expressing, Two Tailed Test, MANN-WHITNEY, One-tailed Test

    SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. A Representative multi-color immunofluorescence image of CD3, PD-1, CD20, Bcl-6 with DAPI staining in mediastinal lymph nodes. Two connecting sections were stained with CD3/PD-1 and CD20/ Bcl-6/CD3 to visualize germinal center (GC) T fh cells and GC B cells, respectively. Images in (a–d) showing GC B cells and images in (f–i) showing GC T fh cells are enlarged from white boxes in (e) and collected using a ×20 objective. Merged image in (d) shows CD20+Bcl-6+ GC B cells and image in (i) shows CD3 + PD-1+ GC T fh cells. Scale bar in (e) is 100 µm and the rest are 25 µm. CD3 stain in pink is pseudo color (original red) to distinguish from Bcl-6. B Representative gating strategy to identify follicular dendritic cells (FDC), germinal center B cells (GC B), and germinal center T fh cells (GC T fh ) in the mediastinal lymph nodes (Med) Fluorochromes used were CD45-A488, CD3-A700, CD20-BV421, Dead-BV510, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe-Cy7, Bcl-6-APC-Cy7, CD140b-APC, CD21-PECF594, CXCR3-BV786. C Median fluorescence intensity of Bcl-6, CD21, CD140b, and CXCR3. D Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (Med, data shown from n = 8 independent animals (GC T fh ; ** p = 0.007, * p = 0.01) relative to cervical lymph nodes (CLN, data shown from n = 8 independent animals) and mesenteric lymph nodes (Mes, data shown from n = 7 independent animals) using a two-tailed Wilcoxon matched-pairs signed-rank test, GC B cells; * p = 0.04 using a two-tailed Wilcoxon matched-pairs signed-rank test, FDCs; p = 0.039 using a two-tailed Wilcoxon matched-pairs signed-rank test. Horizontal line indicates median. E Majority of GC T fh cells in mediastinal lymph nodes express CXCR3 (GC T fh and T fh ; ** p = 0.007, * p = 0.01, and mTfh * p = 0.01 relative to CLN and Mes using a two-tailed Wilcoxon matched-pairs signed-rank test). Data shown are from n = 8 independent animals for Med, CLN, and n = 7 independent animals for Mes. Horizontal line indicates median.
    Figure Legend Snippet: SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. A Representative multi-color immunofluorescence image of CD3, PD-1, CD20, Bcl-6 with DAPI staining in mediastinal lymph nodes. Two connecting sections were stained with CD3/PD-1 and CD20/ Bcl-6/CD3 to visualize germinal center (GC) T fh cells and GC B cells, respectively. Images in (a–d) showing GC B cells and images in (f–i) showing GC T fh cells are enlarged from white boxes in (e) and collected using a ×20 objective. Merged image in (d) shows CD20+Bcl-6+ GC B cells and image in (i) shows CD3 + PD-1+ GC T fh cells. Scale bar in (e) is 100 µm and the rest are 25 µm. CD3 stain in pink is pseudo color (original red) to distinguish from Bcl-6. B Representative gating strategy to identify follicular dendritic cells (FDC), germinal center B cells (GC B), and germinal center T fh cells (GC T fh ) in the mediastinal lymph nodes (Med) Fluorochromes used were CD45-A488, CD3-A700, CD20-BV421, Dead-BV510, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe-Cy7, Bcl-6-APC-Cy7, CD140b-APC, CD21-PECF594, CXCR3-BV786. C Median fluorescence intensity of Bcl-6, CD21, CD140b, and CXCR3. D Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (Med, data shown from n = 8 independent animals (GC T fh ; ** p = 0.007, * p = 0.01) relative to cervical lymph nodes (CLN, data shown from n = 8 independent animals) and mesenteric lymph nodes (Mes, data shown from n = 7 independent animals) using a two-tailed Wilcoxon matched-pairs signed-rank test, GC B cells; * p = 0.04 using a two-tailed Wilcoxon matched-pairs signed-rank test, FDCs; p = 0.039 using a two-tailed Wilcoxon matched-pairs signed-rank test. Horizontal line indicates median. E Majority of GC T fh cells in mediastinal lymph nodes express CXCR3 (GC T fh and T fh ; ** p = 0.007, * p = 0.01, and mTfh * p = 0.01 relative to CLN and Mes using a two-tailed Wilcoxon matched-pairs signed-rank test). Data shown are from n = 8 independent animals for Med, CLN, and n = 7 independent animals for Mes. Horizontal line indicates median.

    Techniques Used: Infection, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

    SARS-CoV-2 infection leads to rapid and transient shifts in innate immune responses in peripheral blood. A Representative gating strategy for innate immune subsets in whole blood after gating on singlets. Fluorochromes used were CD3/CD20- APC-Cy7, CD14-A700, CD8- BUV 805, CD66-APC, HLA-DR-BV786, CD16-BV605, CD123-BV421, CD11c-Pe-Cy7. B Kinetics of innate immune responses (pro-inflammatory monocytes; * p = 0.01 at d2 and d4 relative to d0 using a one-tailed paired t test in infected animals, ** p = 0.006 and 0.002 at d2 and d4 relative to d0 in infused animals, pDCs; ** p = 0.005 at d2 relative to d0 using a one-tailed paired t test in infected animals, * p = 0.01 at d2 relative to d0 using a one-tailed paired t test in infused animals, mDCs; * p = 0.02 at d2 relative to d0 using a one-tailed paired t test in infected animals). C Serum chemokines monocyte chemoattractant protein (MCP)-1, interferon gamma induced protein (IP)-10, and interferon induced T-cell alpha chemoattractant (I-TAC) (MCP-1; ** p = 0.001 for infected and ** p = 0.005 for infused at d2 relative to d0 using a one-tailed paired t test, IP-10; * p = 0.03 for infected and *** p = 0.0008 for infused at d2 relative to d0 using a one-tailed paired t test, ITAC; *** p = 0.0005 for infected and *** p = 0.0007 for infused at d2 relative to d0 using a one-tailed paired t test). D Correlation of innate immune cells against chemokines, and interleukin (IL)-10 vs IL-6 (two-tailed Pearson test p values shown, 95% confidence bands of the best fit line are shown).
    Figure Legend Snippet: SARS-CoV-2 infection leads to rapid and transient shifts in innate immune responses in peripheral blood. A Representative gating strategy for innate immune subsets in whole blood after gating on singlets. Fluorochromes used were CD3/CD20- APC-Cy7, CD14-A700, CD8- BUV 805, CD66-APC, HLA-DR-BV786, CD16-BV605, CD123-BV421, CD11c-Pe-Cy7. B Kinetics of innate immune responses (pro-inflammatory monocytes; * p = 0.01 at d2 and d4 relative to d0 using a one-tailed paired t test in infected animals, ** p = 0.006 and 0.002 at d2 and d4 relative to d0 in infused animals, pDCs; ** p = 0.005 at d2 relative to d0 using a one-tailed paired t test in infected animals, * p = 0.01 at d2 relative to d0 using a one-tailed paired t test in infused animals, mDCs; * p = 0.02 at d2 relative to d0 using a one-tailed paired t test in infected animals). C Serum chemokines monocyte chemoattractant protein (MCP)-1, interferon gamma induced protein (IP)-10, and interferon induced T-cell alpha chemoattractant (I-TAC) (MCP-1; ** p = 0.001 for infected and ** p = 0.005 for infused at d2 relative to d0 using a one-tailed paired t test, IP-10; * p = 0.03 for infected and *** p = 0.0008 for infused at d2 relative to d0 using a one-tailed paired t test, ITAC; *** p = 0.0005 for infected and *** p = 0.0007 for infused at d2 relative to d0 using a one-tailed paired t test). D Correlation of innate immune cells against chemokines, and interleukin (IL)-10 vs IL-6 (two-tailed Pearson test p values shown, 95% confidence bands of the best fit line are shown).

    Techniques Used: Infection, One-tailed Test, Two Tailed Test

    CD4 T fh cells targeting spike (S) and nucleocapsid (N) are generated in lymphoid tissue following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with peptide megapools; membrane (M), open reading frame non-structural proteins (ORF-nsp) and Phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Iono) Fluorochromes used were CD3-A700, Dead-APC-Cy7, CD8-BV510, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, CD25-APC, OX40-BV786, IFNG-Pe-Cy7, TNFa-A488, IL-17-BV421, IL-21-APC. B Scatter plot showing Activation-induced marker (AIM) + CD4 subsets. Dashed line represents undetectable responses assigned a value of 0.01% C Gating strategy to identify cytokine profiles (interferon (IFN)γ, interleukin (IL)-2, tumor necrosis factor (TNF)a, interleukin (IL)-17, interleukin (IL)-21) of CXCR5 + , CXCR5-, and CD8 + CD95 + T cells) in spleen following stimulation. D Pie chart shows T-cell polyfunctionality.
    Figure Legend Snippet: CD4 T fh cells targeting spike (S) and nucleocapsid (N) are generated in lymphoid tissue following SARS-CoV-2 infection. A Representative gating strategy to identify SARS-CoV-2-specific CD4 T cells following stimulation with peptide megapools; membrane (M), open reading frame non-structural proteins (ORF-nsp) and Phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Iono) Fluorochromes used were CD3-A700, Dead-APC-Cy7, CD8-BV510, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, CD25-APC, OX40-BV786, IFNG-Pe-Cy7, TNFa-A488, IL-17-BV421, IL-21-APC. B Scatter plot showing Activation-induced marker (AIM) + CD4 subsets. Dashed line represents undetectable responses assigned a value of 0.01% C Gating strategy to identify cytokine profiles (interferon (IFN)γ, interleukin (IL)-2, tumor necrosis factor (TNF)a, interleukin (IL)-17, interleukin (IL)-21) of CXCR5 + , CXCR5-, and CD8 + CD95 + T cells) in spleen following stimulation. D Pie chart shows T-cell polyfunctionality.

    Techniques Used: Generated, Infection, Activation Assay, Marker

    SARS-CoV-2 infection increases the number CD4 T follicular helper cells in peripheral blood. A Representative gating strategy to capture CD4 T cells expressing Ki-67 and programmed death-1 (PD-1) in whole blood. Fluorochromes used were CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, Ki-67-A488, CXCR3-BV786, CCR6-PECF594, CCR4-BV605, SLAM-A488, CX3CR1-PECF594, CD28-Pe-Cy7, CCR7-BV711, ICOS-BV786. B Kinetics show frequency and absolute counts of Ki-67 + PD-1 + CD4 T follicular helper cells (T fh ) cells (% of T fh cells; * p = 0.01 at d4 and d7 relative to d0 for infected and ** p = 0.002 at d7 relative to d0 for infused using a one-tailed paired t test, absolute T fh cell counts; ** p = 0.003 at d4 and ** p = 0.0086 at d7 relative to d0 for infected and ** p = 0.003 at d7 relative to d0 for infused using a one-tailed paired t test. Data are from a real-time longitudinal staining of whole blood performed a single time) C correlation plots of Ki-67 + CD8 T cells against Ki-67 + CD4 subsets, and viral(v)RNA (all day 7) (two-tailed Pearson test p values shown. 95% confidence bands of the best fit line are shown) D t-distributed stochastic neighbor embedding (tSNE) plot based on flow cytometry data of CD4 Ki-67 + events at Day 7 from infected (16,197 events) and infected + infused animals (22,406 events); dot plot shows frequency of Ki-67 + CD4 T-cell subsets. ( E – F ) Histograms and median fluorescence intensity (MFI) dot plots illustrate relative expression of signaling lymphocyte activation molecule (SLAM), CX3C chemokine receptor 1 (CX3CR1), CD28, and C-C chemokine receptor type 7(CCR7) within four different populations identified at Day 7 in peripheral blood mononuclear cells (PBMCs, n = 7). Unique symbols identify animals in each of the experimental groups.
    Figure Legend Snippet: SARS-CoV-2 infection increases the number CD4 T follicular helper cells in peripheral blood. A Representative gating strategy to capture CD4 T cells expressing Ki-67 and programmed death-1 (PD-1) in whole blood. Fluorochromes used were CD3-A700, CD20/Dead-APC-Cy7, CD8-BUV 805, CD4-BV650, CD95-BUV737, CXCR5-PE, PD-1-Pe Cy7, Ki-67-A488, CXCR3-BV786, CCR6-PECF594, CCR4-BV605, SLAM-A488, CX3CR1-PECF594, CD28-Pe-Cy7, CCR7-BV711, ICOS-BV786. B Kinetics show frequency and absolute counts of Ki-67 + PD-1 + CD4 T follicular helper cells (T fh ) cells (% of T fh cells; * p = 0.01 at d4 and d7 relative to d0 for infected and ** p = 0.002 at d7 relative to d0 for infused using a one-tailed paired t test, absolute T fh cell counts; ** p = 0.003 at d4 and ** p = 0.0086 at d7 relative to d0 for infected and ** p = 0.003 at d7 relative to d0 for infused using a one-tailed paired t test. Data are from a real-time longitudinal staining of whole blood performed a single time) C correlation plots of Ki-67 + CD8 T cells against Ki-67 + CD4 subsets, and viral(v)RNA (all day 7) (two-tailed Pearson test p values shown. 95% confidence bands of the best fit line are shown) D t-distributed stochastic neighbor embedding (tSNE) plot based on flow cytometry data of CD4 Ki-67 + events at Day 7 from infected (16,197 events) and infected + infused animals (22,406 events); dot plot shows frequency of Ki-67 + CD4 T-cell subsets. ( E – F ) Histograms and median fluorescence intensity (MFI) dot plots illustrate relative expression of signaling lymphocyte activation molecule (SLAM), CX3C chemokine receptor 1 (CX3CR1), CD28, and C-C chemokine receptor type 7(CCR7) within four different populations identified at Day 7 in peripheral blood mononuclear cells (PBMCs, n = 7). Unique symbols identify animals in each of the experimental groups.

    Techniques Used: Infection, Expressing, One-tailed Test, Staining, Two Tailed Test, Flow Cytometry, Fluorescence, Activation Assay

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Standard Deviation

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

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    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

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

    16) Product Images from "SARS-CoV-2 induces activation and diversification of human plasmacytoid pre-dendritic cells"

    Article Title: SARS-CoV-2 induces activation and diversification of human plasmacytoid pre-dendritic cells

    Journal: bioRxiv

    doi: 10.1101/2020.07.10.197343

    SARS-CoV-2-induced pDC activation is inhibited by hydroxychloroquine. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI 1 with or without the presence of hydroxychloroquine (HCQ). (A) Dotplot showing pDC diversification in P1-, P2-, and P3-subpopulations in the presence of HCQ. (B) Quantification of the three populations. (C) Histograms of pDC’s activation markers. (D) Geometric mean (MFI) of activation markers. Histograms represent medians and bars interquartile of n=3 healthy donors. (E) Quantification of pro-inflammatory cytokines production. Bars represent medians of n=3 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2-induced pDC activation is inhibited by hydroxychloroquine. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI 1 with or without the presence of hydroxychloroquine (HCQ). (A) Dotplot showing pDC diversification in P1-, P2-, and P3-subpopulations in the presence of HCQ. (B) Quantification of the three populations. (C) Histograms of pDC’s activation markers. (D) Geometric mean (MFI) of activation markers. Histograms represent medians and bars interquartile of n=3 healthy donors. (E) Quantification of pro-inflammatory cytokines production. Bars represent medians of n=3 healthy donors. *P

    Techniques Used: Activation Assay, Cell Culture

    SARS-CoV-2-activated pDC produce pro-inflammatory cytokines. Sorted blood pDC from healthy donors were cultured for 24h or 48h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 1. (A) Quantification of pro-inflammatory cytokines at 24h. Bars represent medians of n=5 healthy donors. (B) Quantification of pro-inflammatory cytokines at 48h. Bars represent medians of n=3 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2-activated pDC produce pro-inflammatory cytokines. Sorted blood pDC from healthy donors were cultured for 24h or 48h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 1. (A) Quantification of pro-inflammatory cytokines at 24h. Bars represent medians of n=5 healthy donors. (B) Quantification of pro-inflammatory cytokines at 48h. Bars represent medians of n=3 healthy donors. *P

    Techniques Used: Cell Culture

    SARS-CoV-2 induces activation and diversification of primary human pDC. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, SARS-CoV-2, or Influenza virus A (Flu). (A) Dotplot showing pDC activation and diversification through the expression of PD-L1 and CD80 into P1-, P2-, and P3-subpopulations. (B) Quantification of the three populations. Bars represent medians of n=5 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2 induces activation and diversification of primary human pDC. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, SARS-CoV-2, or Influenza virus A (Flu). (A) Dotplot showing pDC activation and diversification through the expression of PD-L1 and CD80 into P1-, P2-, and P3-subpopulations. (B) Quantification of the three populations. Bars represent medians of n=5 healthy donors. *P

    Techniques Used: Activation Assay, Cell Culture, Expressing

    SARS-CoV-2 induces pDC activation in a dose dependent manner. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 0.04, 0.2, or 1. (A) Dotplot showing pDC activation through the expression of PD-L1 and CD80. (B) Quantification of the three populations. Bars represent medians of n=3 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2 induces pDC activation in a dose dependent manner. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 0.04, 0.2, or 1. (A) Dotplot showing pDC activation through the expression of PD-L1 and CD80. (B) Quantification of the three populations. Bars represent medians of n=3 healthy donors. *P

    Techniques Used: Activation Assay, Cell Culture, Expressing

    17) Product Images from "Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors"

    Article Title: Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

    Journal: bioRxiv

    doi: 10.1101/2020.07.22.215236

    Establishment of the CSBT and CRBT assays. (A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2-overexpressing cell lines. EF1αp, human EF-1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B-fused mRuby3; BsR, blasticidin S-resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2-mRb3, human ACE2 with C-terminal fusing of mRuby3; H2BiRFP, H2B-fused iRFP670; PuR, puromycin resistance gene. (B) Western blot analyses of expressions of ACE2 and TMPRSS2 in 293T and H1299 cells stably transfected with different constructs. NT cell, non-transfected cells. (C) Fluorescence confocal images of 293T-ACE2iRb3 cells incubated with SARS-CoV2-RBG and SARS-CoV2-STG for different times. The nucleus H2B-iRFP670 was pseudo-colored blue. The scale bar was 10 μm. (D) Schematic illustration of the procedures of cell-based high-content imaging assay using fluorescent RBG or STG viral entry sensors. (E) Dose-dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T-ACE2iRb3 cells. SARS-CoV2-RBD488 was a dylight488-conjugated SARS-CoV2-RBD protein, and SARS-CoV2-ST488 was a dylight488-conjugated SARS-CoV2-ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 nM, respectively. (F) Comparisons of the fluorescence response (cMFI) of various SARS-CoV-2 probes on 293T-ACE2iRb3 cells. For panel E and F, cell images were obtained for 25 different views for each test, and the data were expressed as mean±SD. (G) Dose-dependent cMFI inhibition of recombinant ACE2, SARS-CoV2-RBD, and SARS-CoV2-S1 proteins for the binding and uptake of SARS-CoV2-STG (upper panel) and SARS-CoV2-RBG (lower panel). The experiments were performed following the procedure as described in panel D. The data were mean±SD. CSBT, cell-based spike function blocking test; CRBT, cell-based RBD function blocking test.
    Figure Legend Snippet: Establishment of the CSBT and CRBT assays. (A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2-overexpressing cell lines. EF1αp, human EF-1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B-fused mRuby3; BsR, blasticidin S-resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2-mRb3, human ACE2 with C-terminal fusing of mRuby3; H2BiRFP, H2B-fused iRFP670; PuR, puromycin resistance gene. (B) Western blot analyses of expressions of ACE2 and TMPRSS2 in 293T and H1299 cells stably transfected with different constructs. NT cell, non-transfected cells. (C) Fluorescence confocal images of 293T-ACE2iRb3 cells incubated with SARS-CoV2-RBG and SARS-CoV2-STG for different times. The nucleus H2B-iRFP670 was pseudo-colored blue. The scale bar was 10 μm. (D) Schematic illustration of the procedures of cell-based high-content imaging assay using fluorescent RBG or STG viral entry sensors. (E) Dose-dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T-ACE2iRb3 cells. SARS-CoV2-RBD488 was a dylight488-conjugated SARS-CoV2-RBD protein, and SARS-CoV2-ST488 was a dylight488-conjugated SARS-CoV2-ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 nM, respectively. (F) Comparisons of the fluorescence response (cMFI) of various SARS-CoV-2 probes on 293T-ACE2iRb3 cells. For panel E and F, cell images were obtained for 25 different views for each test, and the data were expressed as mean±SD. (G) Dose-dependent cMFI inhibition of recombinant ACE2, SARS-CoV2-RBD, and SARS-CoV2-S1 proteins for the binding and uptake of SARS-CoV2-STG (upper panel) and SARS-CoV2-RBG (lower panel). The experiments were performed following the procedure as described in panel D. The data were mean±SD. CSBT, cell-based spike function blocking test; CRBT, cell-based RBD function blocking test.

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

    The 83H7 mAb inhibits SARS-CoV-2 via the intracellular neutralization pathway. The 293T-ACE2iRb3 cells were incubated with 20 nM of dylight633-labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 nM). Live-cell fluorescence image dynamically tracked using a 63x water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time-series (at 10-min, 1-hour, 2-hour, 3-hour, 5-hour, 7-hour, 9-hour, 11-hour, and 13-hour) analyses of the STG-IVNs (A), STG-IVpMFI (B), Ab633-IVNs (C), Ab633-IVpMFI (D) and the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles (E). IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. (F) Comparisons of the STG-IVA of the internalized STG vesicles among groups co-incubated with various mAbs at 5-hour post-incubation. ** indicates p
    Figure Legend Snippet: The 83H7 mAb inhibits SARS-CoV-2 via the intracellular neutralization pathway. The 293T-ACE2iRb3 cells were incubated with 20 nM of dylight633-labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 nM). Live-cell fluorescence image dynamically tracked using a 63x water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time-series (at 10-min, 1-hour, 2-hour, 3-hour, 5-hour, 7-hour, 9-hour, 11-hour, and 13-hour) analyses of the STG-IVNs (A), STG-IVpMFI (B), Ab633-IVNs (C), Ab633-IVpMFI (D) and the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles (E). IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. (F) Comparisons of the STG-IVA of the internalized STG vesicles among groups co-incubated with various mAbs at 5-hour post-incubation. ** indicates p

    Techniques Used: Neutralization, Incubation, Labeling, Fluorescence

    Evaluation of neutralization potential of human plasmas from convalescent COVID-19 patients by CSBT and CRBT assays. (A) Comparisons of cMFI inhibitions on CSBT and CRBT assays between plasma samples from convalescent COVID-19 patients and healthy control (HC) subjects. The cMFI inhibition (%) at 1:20 dilution was plotted at the left Y-axis. The cutoff values for CSBT and CRBT were inhibition of 25% (median HC value +3.3×SD) on cMFI at 1:20 dilution. (B) Heatmaps showing CSBT and CRBT effects of two-fold serial dilutions of 32 plasmas from convalescent COVID-19 patients. (C) Distributions of the levels of TAb, IgM, IgG, CSBT, CRBT and LVppNAT of convalescent plasma samples. The numbers indicated the average titers at log10. The titers of Ab, IgM, and IgG were expressed as relative S/CO values determined by serial dilution measurements of each sample (maximum reactive dilution fold multiplied by S/CO). The CRBT and CSBT titers were expresses at ID25, whereas the LVppNAT was expressed as ID50. (D) Correlation analyses between the CSBT titer and the CRBT efficiency (at 1:20 dilution), the TAb titer, the IgM titer, the IgG titer, the LVppNAT and the NAT against authentic SARS-CoV-2 virus among convalescent plasmas. The correlation of CSBT titer and neutralization activity against authentic SARS-CoV-2 virus in 12 representative samples (included 11 convalescent COVID-19 plasmas and 1 control sample).
    Figure Legend Snippet: Evaluation of neutralization potential of human plasmas from convalescent COVID-19 patients by CSBT and CRBT assays. (A) Comparisons of cMFI inhibitions on CSBT and CRBT assays between plasma samples from convalescent COVID-19 patients and healthy control (HC) subjects. The cMFI inhibition (%) at 1:20 dilution was plotted at the left Y-axis. The cutoff values for CSBT and CRBT were inhibition of 25% (median HC value +3.3×SD) on cMFI at 1:20 dilution. (B) Heatmaps showing CSBT and CRBT effects of two-fold serial dilutions of 32 plasmas from convalescent COVID-19 patients. (C) Distributions of the levels of TAb, IgM, IgG, CSBT, CRBT and LVppNAT of convalescent plasma samples. The numbers indicated the average titers at log10. The titers of Ab, IgM, and IgG were expressed as relative S/CO values determined by serial dilution measurements of each sample (maximum reactive dilution fold multiplied by S/CO). The CRBT and CSBT titers were expresses at ID25, whereas the LVppNAT was expressed as ID50. (D) Correlation analyses between the CSBT titer and the CRBT efficiency (at 1:20 dilution), the TAb titer, the IgM titer, the IgG titer, the LVppNAT and the NAT against authentic SARS-CoV-2 virus among convalescent plasmas. The correlation of CSBT titer and neutralization activity against authentic SARS-CoV-2 virus in 12 representative samples (included 11 convalescent COVID-19 plasmas and 1 control sample).

    Techniques Used: Neutralization, Inhibition, Serial Dilution, Activity Assay

    Generation and characterization of FP-fused SARS-CoV-2 S proteins. (A) Schematics of STG and RBG constructs. Functional domains are colored. NTD, N-terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; TM, transmembrane domain; cyt, cytoplasmic tail; TFd, T4 fibritin trimerization motif; mGam, monomeric Gamillus; mNG, mNeonGreen. (B) SDS-PAGE and fluorescence analyses for purified ST-based and RBD-based SARS-CoV-2 S proteins. (C) Size-exclusion chromatogram (SEC) of the purified SARS-CoV2-ST, SARS-CoV2-STG and SARS-CoV2-STN. Data from UV280 detector (upper panel) and fluorescence detector (lower panel) from a G3000 HPLC Column were showed. The molecular weight of SARS-CoV2-STG (or SARS-CoV2-STN) was about 808 kd, which was calculated according to its elution time in referring to the standard curve of determining the molecular weight as shown in Figure S2A and S2B. (D) SPR sensorgrams showing the binding kinetics for SARS-CoV2-STG (upper panel) or SARS-CoV2-RBG (lower panel) with immobilized rACE2 (human). Colored lines represented a global fit of the data using a 1:1 binding model.
    Figure Legend Snippet: Generation and characterization of FP-fused SARS-CoV-2 S proteins. (A) Schematics of STG and RBG constructs. Functional domains are colored. NTD, N-terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; TM, transmembrane domain; cyt, cytoplasmic tail; TFd, T4 fibritin trimerization motif; mGam, monomeric Gamillus; mNG, mNeonGreen. (B) SDS-PAGE and fluorescence analyses for purified ST-based and RBD-based SARS-CoV-2 S proteins. (C) Size-exclusion chromatogram (SEC) of the purified SARS-CoV2-ST, SARS-CoV2-STG and SARS-CoV2-STN. Data from UV280 detector (upper panel) and fluorescence detector (lower panel) from a G3000 HPLC Column were showed. The molecular weight of SARS-CoV2-STG (or SARS-CoV2-STN) was about 808 kd, which was calculated according to its elution time in referring to the standard curve of determining the molecular weight as shown in Figure S2A and S2B. (D) SPR sensorgrams showing the binding kinetics for SARS-CoV2-STG (upper panel) or SARS-CoV2-RBG (lower panel) with immobilized rACE2 (human). Colored lines represented a global fit of the data using a 1:1 binding model.

    Techniques Used: Construct, Functional Assay, Binding Assay, SDS Page, Fluorescence, Purification, High Performance Liquid Chromatography, Molecular Weight, SPR Assay

    Detection of compound-induced influence on SARS-CoV-2 S-mediated cellular entry. (A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. (B) Dose-dependent inhibitions of 11 compounds against SARS-CoV-2 LVpp infection on H1299-ACE2hR cells. All compounds were tested in a 2-fold dilution series, and the initial drug concentrations were begun at their maximal non-cytotoxic concentrations. The initial concentrations were 200 μM for amiloride, MDC and DMSO (as a solvent control); 100 μM for dynasore; 10 μM for filipin, APY0201, YM201636 and tetrandrine; 4 μM for nystatin; 100 nM for Baf.A1 and apilimod. ND, not detected. (C) Confocal images of STG (green channel), ACE2-mRuby3 (red channel), and nucleus (blue channel) in 293T-ACE2iRb3 cells at 5-hour post STG incubation. The cells were pretreated with compounds for 1-hour before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above-mentioned. Scale bar, 10 μm. (D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose-dependent influence of various compounds on STG internalization characteristics on 293T-ACE2iRb3 cells at 1-hour (left panels) and 5-hour (right panels) after incubation. All compounds were tested in a 4-fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left-to-right orderly displayed the values measured from cells treated with 4-fold serial high-to-low concentrations of compounds. STG-IFR, internalized STG fluorescence intensity ratio; STG-IVA, average area (μm 2 ) of internalized STG vesicles; STG-IVNs, average numbers of internalized STG vesicles per cell; *, p
    Figure Legend Snippet: Detection of compound-induced influence on SARS-CoV-2 S-mediated cellular entry. (A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. (B) Dose-dependent inhibitions of 11 compounds against SARS-CoV-2 LVpp infection on H1299-ACE2hR cells. All compounds were tested in a 2-fold dilution series, and the initial drug concentrations were begun at their maximal non-cytotoxic concentrations. The initial concentrations were 200 μM for amiloride, MDC and DMSO (as a solvent control); 100 μM for dynasore; 10 μM for filipin, APY0201, YM201636 and tetrandrine; 4 μM for nystatin; 100 nM for Baf.A1 and apilimod. ND, not detected. (C) Confocal images of STG (green channel), ACE2-mRuby3 (red channel), and nucleus (blue channel) in 293T-ACE2iRb3 cells at 5-hour post STG incubation. The cells were pretreated with compounds for 1-hour before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above-mentioned. Scale bar, 10 μm. (D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose-dependent influence of various compounds on STG internalization characteristics on 293T-ACE2iRb3 cells at 1-hour (left panels) and 5-hour (right panels) after incubation. All compounds were tested in a 4-fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left-to-right orderly displayed the values measured from cells treated with 4-fold serial high-to-low concentrations of compounds. STG-IFR, internalized STG fluorescence intensity ratio; STG-IVA, average area (μm 2 ) of internalized STG vesicles; STG-IVNs, average numbers of internalized STG vesicles per cell; *, p

    Techniques Used: Infection, Incubation, Confocal Microscopy, Fluorescence

    18) Product Images from "Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species"

    Article Title: Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153916

    Susceptibility to SARS-CoV-2 of HEK293T cells conferred by different species of ACE2. HEK293T cells were transfected with plasmids expressing indicated ACE2. Cells were infected with 0.5 MOI of SARS-CoV-2 24 h after the transfection, and were detected for the replication of SARS-CoV-2 by IFA.
    Figure Legend Snippet: Susceptibility to SARS-CoV-2 of HEK293T cells conferred by different species of ACE2. HEK293T cells were transfected with plasmids expressing indicated ACE2. Cells were infected with 0.5 MOI of SARS-CoV-2 24 h after the transfection, and were detected for the replication of SARS-CoV-2 by IFA.

    Techniques Used: Transfection, Expressing, Infection, Immunofluorescence

    Sequence composition of the Rhesus monkeyACE2 cloned in this study with that of the prototype monkey ACE2 and susceptibility to SARS-CoV-2 of 217 restoration. ( A )Two sites of natural variation (R192G and Y217N) were identified in the cDNA of Rhesus monkey ACE cloned in this study were compared with the monkey prototype ACE and the human ACE. ( B ) HEK293T cells were transfected with plasmids expressing indicated ACE2. Cells were infected with 0.5 MOI of SARS-CoV-2 24 h after the transfection, and were detected for the replication of SARS-CoV-2 by IFA.
    Figure Legend Snippet: Sequence composition of the Rhesus monkeyACE2 cloned in this study with that of the prototype monkey ACE2 and susceptibility to SARS-CoV-2 of 217 restoration. ( A )Two sites of natural variation (R192G and Y217N) were identified in the cDNA of Rhesus monkey ACE cloned in this study were compared with the monkey prototype ACE and the human ACE. ( B ) HEK293T cells were transfected with plasmids expressing indicated ACE2. Cells were infected with 0.5 MOI of SARS-CoV-2 24 h after the transfection, and were detected for the replication of SARS-CoV-2 by IFA.

    Techniques Used: Sequencing, Clone Assay, Transfection, Expressing, Infection, Immunofluorescence

    19) Product Images from "Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals"

    Article Title: Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals

    Journal: bioRxiv

    doi: 10.1101/2020.05.13.092619

    Neutralization of SARS-CoV-2 pseudovirus by plasma.
    Figure Legend Snippet: Neutralization of SARS-CoV-2 pseudovirus by plasma.

    Techniques Used: Neutralization

    Anti-SARS-CoV-2 RBD antibodies.
    Figure Legend Snippet: Anti-SARS-CoV-2 RBD antibodies.

    Techniques Used:

    Diagrammatic representation of the SARS-CoV2 pseudovirus luciferase assay. a , Co-transfection of pNL4-3ΔEnv-nanoluc and pSARS-CoV-2 spike vectors into 293T cells leads to production of SARS-CoV-2 Spike-pseudotyped HIV-1 particles (SARS-CoV-2 pseudovirus) carrying the Nanoluc gene. b , SARS-CoV-2 pseudovirus is incubated for 1 h at 37°C with plasma or monoclonal antibody dilutions. The virus-antibody mixture is used to infect ACE2-expressing 293T cells, which will express nanoluc Luciferase upon infection. c, Relative luminescence units (RLU) reads from lysates of ACE2-expressing 293T cells infected with increasing amounts of SARS-CoV-2 pseudovirus.
    Figure Legend Snippet: Diagrammatic representation of the SARS-CoV2 pseudovirus luciferase assay. a , Co-transfection of pNL4-3ΔEnv-nanoluc and pSARS-CoV-2 spike vectors into 293T cells leads to production of SARS-CoV-2 Spike-pseudotyped HIV-1 particles (SARS-CoV-2 pseudovirus) carrying the Nanoluc gene. b , SARS-CoV-2 pseudovirus is incubated for 1 h at 37°C with plasma or monoclonal antibody dilutions. The virus-antibody mixture is used to infect ACE2-expressing 293T cells, which will express nanoluc Luciferase upon infection. c, Relative luminescence units (RLU) reads from lysates of ACE2-expressing 293T cells infected with increasing amounts of SARS-CoV-2 pseudovirus.

    Techniques Used: Luciferase, Cotransfection, Incubation, Expressing, Infection

    Anti-SARS-CoV-2 RBD antibody reactivity.
    Figure Legend Snippet: Anti-SARS-CoV-2 RBD antibody reactivity.

    Techniques Used:

    Plasma antibodies against SARS-CoV-2.
    Figure Legend Snippet: Plasma antibodies against SARS-CoV-2.

    Techniques Used:

    Binding of the monoclonal antibodies to the RBD of SARS-CoV-2 and SARS-CoV. a , EC 50 values for binding to the RBD of SARS-CoV-2. b and c , Binding curves and EC 50 values for binding to the RBD of SARS-CoV.
    Figure Legend Snippet: Binding of the monoclonal antibodies to the RBD of SARS-CoV-2 and SARS-CoV. a , EC 50 values for binding to the RBD of SARS-CoV-2. b and c , Binding curves and EC 50 values for binding to the RBD of SARS-CoV.

    Techniques Used: Binding Assay

    Frequency distributions of human V genes. The two-tailed t test with unequal variance was used to compare the frequency distributions of human V genes of anti-SARS-CoV-2 antibodies from this study to Sequence Read Archive SRP010970 34 .
    Figure Legend Snippet: Frequency distributions of human V genes. The two-tailed t test with unequal variance was used to compare the frequency distributions of human V genes of anti-SARS-CoV-2 antibodies from this study to Sequence Read Archive SRP010970 34 .

    Techniques Used: Two Tailed Test, Sequencing

    20) Product Images from "A human monoclonal antibody blocking SARS-CoV-2 infection"

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.987958

    Protein sequence alignment of the S1 B receptor binding in (RBD) of the SARS-CoV and SARS-CoV-2 spike proteins by ClustalW. Numbering denotes the residue position in the full-length spike protein of SARS-CoV (Genbank: AAP13441.1) and SARS-CoV-2 (Genbank: QHD43416.1). Asterisks (*) indicated fully conserved residues, the colon symbol (:) indicates conservation between groups of very similar properties, and the period symbol (.) indicates conservation between groups of weakly similar properties. Sequences corresponding to the S1 B receptor binding core domain and the receptor binding subdomain are colored in blue and orange, respectively. The fourteen residues that are involved in binding of SARS-CoV S1 B human ACE2 are highlighted in grey 1 .
    Figure Legend Snippet: Protein sequence alignment of the S1 B receptor binding in (RBD) of the SARS-CoV and SARS-CoV-2 spike proteins by ClustalW. Numbering denotes the residue position in the full-length spike protein of SARS-CoV (Genbank: AAP13441.1) and SARS-CoV-2 (Genbank: QHD43416.1). Asterisks (*) indicated fully conserved residues, the colon symbol (:) indicates conservation between groups of very similar properties, and the period symbol (.) indicates conservation between groups of weakly similar properties. Sequences corresponding to the S1 B receptor binding core domain and the receptor binding subdomain are colored in blue and orange, respectively. The fourteen residues that are involved in binding of SARS-CoV S1 B human ACE2 are highlighted in grey 1 .

    Techniques Used: Sequencing, Binding Assay

    The neutralizing 47D11 monoclonal antibody binds the receptor binding domain of SARS-CoV and SARS-CoV-2 spike proteins without eliminating S1 B /ACE2 receptor interaction. a) ELISA binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from at least two independent experiments performed is shown. b) Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analysed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Suppl.Fig.4 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analysed for (ACE2-)GFP expression (x-axis) and S1 B binding (y-axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. c) Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 18 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: Similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white.
    Figure Legend Snippet: The neutralizing 47D11 monoclonal antibody binds the receptor binding domain of SARS-CoV and SARS-CoV-2 spike proteins without eliminating S1 B /ACE2 receptor interaction. a) ELISA binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from at least two independent experiments performed is shown. b) Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analysed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Suppl.Fig.4 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analysed for (ACE2-)GFP expression (x-axis) and S1 B binding (y-axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. c) Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 18 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: Similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing

    H2L2 monoclonal antibodies 35F4 and 43C6 neutralize SARS-CoV but not SARS-CoV-2. Antibody-mediated neutralization of infection of VSV particles pseudotyped with spike proteins of SARS-CoV (upper panel) and SARS-CoV-2 (lower panel) by the 35F4 and 43C6 H2L2 antibodies targeting SARS-S1 but not SARS2-S1 (see Suppl.Fig.1 ). An irrelevant antibody was taken along as a human IgG1 isotype control. Means ± SD of triplicates are shown.
    Figure Legend Snippet: H2L2 monoclonal antibodies 35F4 and 43C6 neutralize SARS-CoV but not SARS-CoV-2. Antibody-mediated neutralization of infection of VSV particles pseudotyped with spike proteins of SARS-CoV (upper panel) and SARS-CoV-2 (lower panel) by the 35F4 and 43C6 H2L2 antibodies targeting SARS-S1 but not SARS2-S1 (see Suppl.Fig.1 ). An irrelevant antibody was taken along as a human IgG1 isotype control. Means ± SD of triplicates are shown.

    Techniques Used: Neutralization, Infection

    Binding kinetics of 47D11 to the S ectodomain and S1 B of SARS-CoV and SARS-CoV-2. Binding kinetics of 47D11 to immobilized recombinant SARS-S ecto , SARS2-S ecto , SARS-S1 B and SARS2-S1 B was measured using biolayer interferometry at 25°C, as described previously 21 . Kinetic binding assay was performed by loading 47D11 mAb at optimal concentration (42 nM) on anti-human Fc biosensor for 10 mins. Antigen association step was performed by incubating the sensor with a range of concentrations of the recombinant spike ectodomain (1600-800-400-200-100-50-25 nM) for 10 min, followed by a dissociation step in PBS for 60 min. The kinetics constants were calculated using 1:1 Langmuir binding model on Fortebio Data Analysis 7.0 software.
    Figure Legend Snippet: Binding kinetics of 47D11 to the S ectodomain and S1 B of SARS-CoV and SARS-CoV-2. Binding kinetics of 47D11 to immobilized recombinant SARS-S ecto , SARS2-S ecto , SARS-S1 B and SARS2-S1 B was measured using biolayer interferometry at 25°C, as described previously 21 . Kinetic binding assay was performed by loading 47D11 mAb at optimal concentration (42 nM) on anti-human Fc biosensor for 10 mins. Antigen association step was performed by incubating the sensor with a range of concentrations of the recombinant spike ectodomain (1600-800-400-200-100-50-25 nM) for 10 min, followed by a dissociation step in PBS for 60 min. The kinetics constants were calculated using 1:1 Langmuir binding model on Fortebio Data Analysis 7.0 software.

    Techniques Used: Binding Assay, Recombinant, Concentration Assay, Software

    47D11 neutralizes SARS-CoV and SARS-CoV-2. a) Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b) Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least two independent experiments performed is shown. Iso-CTRL: irrelevant isotype monoclonal antibody. c) Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown.
    Figure Legend Snippet: 47D11 neutralizes SARS-CoV and SARS-CoV-2. a) Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b) Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least two independent experiments performed is shown. Iso-CTRL: irrelevant isotype monoclonal antibody. c) Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown.

    Techniques Used: Binding Assay, Expressing, Immunofluorescence, Negative Control, Neutralization, Infection, Luciferase, Incubation, Transduction

    21) Product Images from "Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate"

    Article Title: Transmission and protection against re-infection in the ferret model with the SARS-CoV-2 USA-WA1/2020 reference isolate

    Journal: bioRxiv

    doi: 10.1101/2020.11.20.392381

    Antibody and viral titers in SARS-CoV-2 infected mock and RBD vaccinated ferrets. Panels A displays binding antibody titers against the S-protein RBD determined by ELISA on days 0, 14, 28, 42, and 56 post-primary vaccination. Red open symbols represent RBD vaccinated ferrets. Closed black symbols represent mock vaccinated animals. Animals were given a secondary vaccination on day 28. Panel B displays neutralizing antibody titers on day 56. Panel C and D display nasal wash titers in mock and RBD vaccinated animals challenged with SARS-CoV-2, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Horizontal dashed line indicates limit of detection.
    Figure Legend Snippet: Antibody and viral titers in SARS-CoV-2 infected mock and RBD vaccinated ferrets. Panels A displays binding antibody titers against the S-protein RBD determined by ELISA on days 0, 14, 28, 42, and 56 post-primary vaccination. Red open symbols represent RBD vaccinated ferrets. Closed black symbols represent mock vaccinated animals. Animals were given a secondary vaccination on day 28. Panel B displays neutralizing antibody titers on day 56. Panel C and D display nasal wash titers in mock and RBD vaccinated animals challenged with SARS-CoV-2, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Horizontal dashed line indicates limit of detection.

    Techniques Used: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    Direct contact and respiratory transmission of the SARS-CoV-2 USA-WA1/2020 isolate in ferrets. Panels A-C, D-F, and G-I display three separate transmission studies. Panels A-C, and D-F each represent a contact transmission study, while panels G-I display data from a respiratory transmission study. Panels A, D, G display nasal wash titers determined by qRT-PCR (left Y-axis) and TCID50 (right Y-axis) for the SARS-CoV-2 inoculated donor animals in each experiment. Line graphs indicate levels of vRNA and bar graphs indicated infectious titers. Panels B, E, and H similarly display nasal wash titers for contact animals. In a given panel, each shaded bar or symbol represents the same animal sampled over multiple time points. Paired donor and contact animals have the same shaded bar or symbol between panels. Panels C, F, and I show neutralizing antibody titers for each donor and contact animal. For all experiments, 4 pairs of ferrets (2 pairs of males and 2 pairs of females) were used, and nasal wash samples were collected every other day. Blood was collected on day 21 post-contact. In the first direct contact transmission study, panels A-C, one direct contact animal was removed due to fighting with its cage mate. Horizontal dashed line indicates limit of detection.
    Figure Legend Snippet: Direct contact and respiratory transmission of the SARS-CoV-2 USA-WA1/2020 isolate in ferrets. Panels A-C, D-F, and G-I display three separate transmission studies. Panels A-C, and D-F each represent a contact transmission study, while panels G-I display data from a respiratory transmission study. Panels A, D, G display nasal wash titers determined by qRT-PCR (left Y-axis) and TCID50 (right Y-axis) for the SARS-CoV-2 inoculated donor animals in each experiment. Line graphs indicate levels of vRNA and bar graphs indicated infectious titers. Panels B, E, and H similarly display nasal wash titers for contact animals. In a given panel, each shaded bar or symbol represents the same animal sampled over multiple time points. Paired donor and contact animals have the same shaded bar or symbol between panels. Panels C, F, and I show neutralizing antibody titers for each donor and contact animal. For all experiments, 4 pairs of ferrets (2 pairs of males and 2 pairs of females) were used, and nasal wash samples were collected every other day. Blood was collected on day 21 post-contact. In the first direct contact transmission study, panels A-C, one direct contact animal was removed due to fighting with its cage mate. Horizontal dashed line indicates limit of detection.

    Techniques Used: Transmission Assay, Quantitative RT-PCR

    Viral and antibody titers in ferrets re-challenged with SARS-CoV-2 on day 28 and 56 post-primary infection. Panel A and B display nasal wash titers in ferrets re-challenged with SARS-CoV-2 on days 28 and 56 post-primary infection, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Panel C displays neutralizing antibody titers prior to primary infection (day 0), at the time of re-challenge (day 28 or 56) and 14 days post re-challenge (days 42 and 70). Horizontal dashed line indicates limit of detection.
    Figure Legend Snippet: Viral and antibody titers in ferrets re-challenged with SARS-CoV-2 on day 28 and 56 post-primary infection. Panel A and B display nasal wash titers in ferrets re-challenged with SARS-CoV-2 on days 28 and 56 post-primary infection, respectively. Line graphs indicate levels of vRNA determined via N2 gene qRT-PCR (left Y-axis) and bar graphs indicated infectious titers (right Y-axis) determined via TCID50 on Vero cells. Panel C displays neutralizing antibody titers prior to primary infection (day 0), at the time of re-challenge (day 28 or 56) and 14 days post re-challenge (days 42 and 70). Horizontal dashed line indicates limit of detection.

    Techniques Used: Infection, Quantitative RT-PCR

    22) Product Images from "Dexamethasone inhibits SARS-CoV-2 spike pseudotyped virus viropexis by binding to ACE2"

    Article Title: Dexamethasone inhibits SARS-CoV-2 spike pseudotyped virus viropexis by binding to ACE2

    Journal: Virology

    doi: 10.1016/j.virol.2020.12.001

    Effect of GCs on the entrance of SARS-CoV-2 spike pseudotyped virus into ACE2 h cells. A. DEX inhibit the entrance of SARS-CoV-2 spike pseudotyped virus into ACE2 h cells; B. DEX had no effect on pseudovirus gene expression. The experiments were repeated three times. Data are presented as mean ± S.D. ** p
    Figure Legend Snippet: Effect of GCs on the entrance of SARS-CoV-2 spike pseudotyped virus into ACE2 h cells. A. DEX inhibit the entrance of SARS-CoV-2 spike pseudotyped virus into ACE2 h cells; B. DEX had no effect on pseudovirus gene expression. The experiments were repeated three times. Data are presented as mean ± S.D. ** p

    Techniques Used: Expressing

    23) Product Images from "Clinical and pathological findings of SARS-CoV-2 infection and concurrent IgA nephropathy: a case report"

    Article Title: Clinical and pathological findings of SARS-CoV-2 infection and concurrent IgA nephropathy: a case report

    Journal: BMC Nephrology

    doi: 10.1186/s12882-020-02163-3

    Renal biopsy findings. a Glomerulus with a fibrocelluar crescent, adjacent acute tubular injury, as well as associated tubular atrophy/interstitial inflammation (PAS stain; original magnification × 200); b Cells debris within the proximal tubular lumen (PAS stain; original magnification × 400); c direct immunofluorescence staining with IgA (original magnification × 400); d Ultrastructure examination reveals mesangial electron-dense deposits (transmission electron microscopy; original magnification × 2500); e Negative immunohistochemistry staining for the S1 spike protein of SARS-CoV-2 (original magnification × 200). PAS: Periodic Acid-Schiff; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2
    Figure Legend Snippet: Renal biopsy findings. a Glomerulus with a fibrocelluar crescent, adjacent acute tubular injury, as well as associated tubular atrophy/interstitial inflammation (PAS stain; original magnification × 200); b Cells debris within the proximal tubular lumen (PAS stain; original magnification × 400); c direct immunofluorescence staining with IgA (original magnification × 400); d Ultrastructure examination reveals mesangial electron-dense deposits (transmission electron microscopy; original magnification × 2500); e Negative immunohistochemistry staining for the S1 spike protein of SARS-CoV-2 (original magnification × 200). PAS: Periodic Acid-Schiff; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2

    Techniques Used: Staining, Immunofluorescence, Transmission Assay, Electron Microscopy, Immunohistochemistry

    24) Product Images from "Clinical and pathological findings of SARS-CoV-2 infection and concurrent IgA nephropathy: a case report"

    Article Title: Clinical and pathological findings of SARS-CoV-2 infection and concurrent IgA nephropathy: a case report

    Journal: BMC Nephrology

    doi: 10.1186/s12882-020-02163-3

    Renal biopsy findings. a Glomerulus with a fibrocelluar crescent, adjacent acute tubular injury, as well as associated tubular atrophy/interstitial inflammation (PAS stain; original magnification × 200); b Cells debris within the proximal tubular lumen (PAS stain; original magnification × 400); c direct immunofluorescence staining with IgA (original magnification × 400); d Ultrastructure examination reveals mesangial electron-dense deposits (transmission electron microscopy; original magnification × 2500); e Negative immunohistochemistry staining for the S1 spike protein of SARS-CoV-2 (original magnification × 200). PAS: Periodic Acid-Schiff; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2
    Figure Legend Snippet: Renal biopsy findings. a Glomerulus with a fibrocelluar crescent, adjacent acute tubular injury, as well as associated tubular atrophy/interstitial inflammation (PAS stain; original magnification × 200); b Cells debris within the proximal tubular lumen (PAS stain; original magnification × 400); c direct immunofluorescence staining with IgA (original magnification × 400); d Ultrastructure examination reveals mesangial electron-dense deposits (transmission electron microscopy; original magnification × 2500); e Negative immunohistochemistry staining for the S1 spike protein of SARS-CoV-2 (original magnification × 200). PAS: Periodic Acid-Schiff; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2

    Techniques Used: Staining, Immunofluorescence, Transmission Assay, Electron Microscopy, Immunohistochemistry

    25) Product Images from "Trophoblast Damage with Acute and Chronic Intervillositis: Disruption of Placental Barrier by SARS-CoV-2"

    Article Title: Trophoblast Damage with Acute and Chronic Intervillositis: Disruption of Placental Barrier by SARS-CoV-2

    Journal: Human Pathology

    doi: 10.1016/j.humpath.2020.12.004

    SARS-CoV-2 qRT-PCR Results in Placental Samples. Reactions for all samples were run in triplicates with consistent results. C t - cycle threshold: reflects the number of PCR cycles before the beginning of the exponential phase of the amplification curve. The lower the C t value, the higher the quantity of the target template in the sample. RP C t - C t values of the control RNase P primer/probe set. C t of included viral controls were also obtained (not shown) and along with the standard curve generated in each reaction were used for viral copy calculations. *FFPE tissue samples were punched out the paraffin block and weighted, after removal of excess paraffin and before RNA extraction.
    Figure Legend Snippet: SARS-CoV-2 qRT-PCR Results in Placental Samples. Reactions for all samples were run in triplicates with consistent results. C t - cycle threshold: reflects the number of PCR cycles before the beginning of the exponential phase of the amplification curve. The lower the C t value, the higher the quantity of the target template in the sample. RP C t - C t values of the control RNase P primer/probe set. C t of included viral controls were also obtained (not shown) and along with the standard curve generated in each reaction were used for viral copy calculations. *FFPE tissue samples were punched out the paraffin block and weighted, after removal of excess paraffin and before RNA extraction.

    Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, Amplification, Generated, Formalin-fixed Paraffin-Embedded, Blocking Assay, RNA Extraction

    Placental Pathology in SARS-CoV-2 Infection. A. Patchy increased villous density, perivillous fibrin deposition and intervillous inflammatory infiltrate; low magnification. H E. B. Intervillositis with mixed inflammatory infiltrate including a significant population of neutrophils (H E), stained with CD15 (insert). C. Cellular changes of STB with “dusky” nuclei with homogenized hyperchromatic chromatin and karyorrhexis. Brisk villous capillaries and lack of villous stromal and vascular apoptosis. H E. D. An advanced stage of STB damage with cytoplasmic clearing and nuclear pycnosis resulting in “ghost” cells appearance. H E. E. Damaged villi with prominent myofibroblasts. A nodular intravillous fibrin deposition. H E. F. Perivillous fibrin precipitated to the injured STB highlighted by PTAH special stain.
    Figure Legend Snippet: Placental Pathology in SARS-CoV-2 Infection. A. Patchy increased villous density, perivillous fibrin deposition and intervillous inflammatory infiltrate; low magnification. H E. B. Intervillositis with mixed inflammatory infiltrate including a significant population of neutrophils (H E), stained with CD15 (insert). C. Cellular changes of STB with “dusky” nuclei with homogenized hyperchromatic chromatin and karyorrhexis. Brisk villous capillaries and lack of villous stromal and vascular apoptosis. H E. D. An advanced stage of STB damage with cytoplasmic clearing and nuclear pycnosis resulting in “ghost” cells appearance. H E. E. Damaged villi with prominent myofibroblasts. A nodular intravillous fibrin deposition. H E. F. Perivillous fibrin precipitated to the injured STB highlighted by PTAH special stain.

    Techniques Used: Infection, Staining

    Identification of SARS-CoV-2 in Placenta. A. Strong diffuse staining of STB with anti-spike protein antibody (mAb 1A9, red chromogen). B. ISH with spike probe: granular binding pattern in STB. C. IHC with anti-nucleocapsid mAb 001 strongly positive in STB, subset of maternal blood cells and weakly positive in the villous stroma. D. IHC with mAb 001: staining of STB and mononuclear cells circulating in maternal vascular spaces (arrowheads). Insert: double staining with mAb (brown chromogen) and CD68 (red chromogen). E. Portion of villous fibroblast identified by peripheral myofilaments (check marks) with numerous round intracytoplasmic particles, measuring 80-100 nm (arrowheads), consistent with virions. An enlarged virion shown (insert). EM. Bar scales shown.
    Figure Legend Snippet: Identification of SARS-CoV-2 in Placenta. A. Strong diffuse staining of STB with anti-spike protein antibody (mAb 1A9, red chromogen). B. ISH with spike probe: granular binding pattern in STB. C. IHC with anti-nucleocapsid mAb 001 strongly positive in STB, subset of maternal blood cells and weakly positive in the villous stroma. D. IHC with mAb 001: staining of STB and mononuclear cells circulating in maternal vascular spaces (arrowheads). Insert: double staining with mAb (brown chromogen) and CD68 (red chromogen). E. Portion of villous fibroblast identified by peripheral myofilaments (check marks) with numerous round intracytoplasmic particles, measuring 80-100 nm (arrowheads), consistent with virions. An enlarged virion shown (insert). EM. Bar scales shown.

    Techniques Used: Staining, In Situ Hybridization, Binding Assay, Immunohistochemistry, Double Staining

    26) Product Images from "Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation"

    Article Title: Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation

    Journal: Nature Structural & Molecular Biology

    doi: 10.1038/s41594-020-0483-8

    Design and validation of SARS-CoV 2P DS S and MERS-CoV 2P DS S. a , Sequence alignment showing the conservation of the residues involved in and adjacent to the designed disulfide bond across human coronavirus S glycoproteins. Residues are highlighted if they are identical in the alignment (black) or conservatively substituted (gray). Residues are numbered according to the SARS-CoV-2 S sequence. Green triangles highlight residues involved in the designed disulfide bond. b , SDS–PAGE analysis of MERS-CoV 2P S and MERS-CoV 2P DS S in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. c , 3D reconstruction in two orthogonal orientations of negatively stained MERS-CoV 2P DS S confirming proper folding of the designed protein construct. d , SDS–PAGE analysis of SARS-CoV 2P S and SARS-CoV 2P DS S in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. e , 3D reconstruction in two orthogonal orientations of negatively stained SARS-CoV 2P DS S confirming proper folding of the designed protein construct. f , g , Binding of various concentrations of the human neutralizing antibodies S309 ( f ) and S304 ( g ) to immobilized SARS-CoV 2P DS S (green) or SARS-CoV 2P S (black). Data are shown as mean and s.d. of n = 2 technical replicates; data are representative of two independent experiments.
    Figure Legend Snippet: Design and validation of SARS-CoV 2P DS S and MERS-CoV 2P DS S. a , Sequence alignment showing the conservation of the residues involved in and adjacent to the designed disulfide bond across human coronavirus S glycoproteins. Residues are highlighted if they are identical in the alignment (black) or conservatively substituted (gray). Residues are numbered according to the SARS-CoV-2 S sequence. Green triangles highlight residues involved in the designed disulfide bond. b , SDS–PAGE analysis of MERS-CoV 2P S and MERS-CoV 2P DS S in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. c , 3D reconstruction in two orthogonal orientations of negatively stained MERS-CoV 2P DS S confirming proper folding of the designed protein construct. d , SDS–PAGE analysis of SARS-CoV 2P S and SARS-CoV 2P DS S in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. e , 3D reconstruction in two orthogonal orientations of negatively stained SARS-CoV 2P DS S confirming proper folding of the designed protein construct. f , g , Binding of various concentrations of the human neutralizing antibodies S309 ( f ) and S304 ( g ) to immobilized SARS-CoV 2P DS S (green) or SARS-CoV 2P S (black). Data are shown as mean and s.d. of n = 2 technical replicates; data are representative of two independent experiments.

    Techniques Used: Sequencing, SDS Page, Staining, Construct, Binding Assay

    CryoEM data processing and validation. a . Local resolution map calculated using cryoSPARC. b-c . Representative electron micrograph (c) and class averages (b) of SARS-CoV-2 2P DS S embedded in vitreous ice. Scale bar: 100 nm. d . Gold-standard Fourier shell correlation curves. The 0.143 cutoff is indicated by an horizontal blue line. e . Particle orientation distribution plot.
    Figure Legend Snippet: CryoEM data processing and validation. a . Local resolution map calculated using cryoSPARC. b-c . Representative electron micrograph (c) and class averages (b) of SARS-CoV-2 2P DS S embedded in vitreous ice. Scale bar: 100 nm. d . Gold-standard Fourier shell correlation curves. The 0.143 cutoff is indicated by an horizontal blue line. e . Particle orientation distribution plot.

    Techniques Used:

    Evaluation of SARS-CoV-2 2P DS S thermal stability and protease resistance. a , b , Electron microscopy analysis of negatively stained SARS-CoV-2 2P S ( a ) and SARS-CoV-2 2P DS S ( b ) incubated for 20 min at 25, 55 and 85 °C. Black arrows highlight particles that appear to be misfolded. Red arrows highlight particles that appear to be in the postfusion conformation. c – e , Binding of human neutralizing antibody S309 to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black) preincubated for 20 min at 25, 55 and 85 °C ( c ), or for 16 h at 4 °C with 1, 10 or 100 µg ml −1 trypsin ( d ) or chymotrypsin ( e ). Graphs show the area under the curve of binding of serially diluted concentrations of the human neutralizing antibody S309; data are shown as mean and s.d. of n = 2 technical replicates, and are representative of one ( d and e ) or two ( c ) independent experiments. Data behind graphs are available in Supplementary Data 1 .
    Figure Legend Snippet: Evaluation of SARS-CoV-2 2P DS S thermal stability and protease resistance. a , b , Electron microscopy analysis of negatively stained SARS-CoV-2 2P S ( a ) and SARS-CoV-2 2P DS S ( b ) incubated for 20 min at 25, 55 and 85 °C. Black arrows highlight particles that appear to be misfolded. Red arrows highlight particles that appear to be in the postfusion conformation. c – e , Binding of human neutralizing antibody S309 to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black) preincubated for 20 min at 25, 55 and 85 °C ( c ), or for 16 h at 4 °C with 1, 10 or 100 µg ml −1 trypsin ( d ) or chymotrypsin ( e ). Graphs show the area under the curve of binding of serially diluted concentrations of the human neutralizing antibody S309; data are shown as mean and s.d. of n = 2 technical replicates, and are representative of one ( d and e ) or two ( c ) independent experiments. Data behind graphs are available in Supplementary Data 1 .

    Techniques Used: Electron Microscopy, Staining, Incubation, Binding Assay

    Structure-based engineering of a SARS-CoV-2 S trimer in the closed conformation. a , b , Cryo-EM structures of SARS-CoV-2 S with one S B receptor-binding domain open ( a , PDB 6VYB ) and one in the closed state ( b , PDB 6VXX ), used as a basis for the design of intermolecular disulfide bonds 18 . c , Pairs of residues mutated to create potential disulfide bonds are shown with dashed black lines between the Cα In panels a – c , each S protomer is colored distinctly. d , SDS–PAGE analysis in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. βME, β-mercaptoethanol. The uncropped image is shown in Supplementary Data 1 . e , f , Electron micrograph of negatively stained SARS-CoV-2 2P DS S confirming proper folding of the designed protein construct ( e ) and representative two-dimensional class averages ( f ).
    Figure Legend Snippet: Structure-based engineering of a SARS-CoV-2 S trimer in the closed conformation. a , b , Cryo-EM structures of SARS-CoV-2 S with one S B receptor-binding domain open ( a , PDB 6VYB ) and one in the closed state ( b , PDB 6VXX ), used as a basis for the design of intermolecular disulfide bonds 18 . c , Pairs of residues mutated to create potential disulfide bonds are shown with dashed black lines between the Cα In panels a – c , each S protomer is colored distinctly. d , SDS–PAGE analysis in reducing and nonreducing conditions showing formation of an intermolecular disulfide bond. βME, β-mercaptoethanol. The uncropped image is shown in Supplementary Data 1 . e , f , Electron micrograph of negatively stained SARS-CoV-2 2P DS S confirming proper folding of the designed protein construct ( e ) and representative two-dimensional class averages ( f ).

    Techniques Used: Binding Assay, SDS Page, Staining, Construct

    Cryo-EM structure of the closed SARS-CoV-2 2P DS S glycoprotein. a , Cryo-EM map of the SARS-CoV-2 2P DS S trimer in the closed conformation at 2.9-Å resolution. b , Ribbon diagram of the SARS-CoV-2 2P DS S trimer atomic model in the same orientation as in panel a . In panels a and b , each S protomer is colored distinctly. Asterisks show the locations of the introduced disulfide bonds. c , Superimposition of the SARS-CoV-2 2P DS S trimer (green) to the coordinates from the 2.8-Å SARS-CoV-2 2P S structure in the closed conformation, PDB 6VXX (ref. 18 ) (black). d , Enlarged view of the designed disulfide bond with the corresponding region of cryo-EM density shown as a blue mesh.
    Figure Legend Snippet: Cryo-EM structure of the closed SARS-CoV-2 2P DS S glycoprotein. a , Cryo-EM map of the SARS-CoV-2 2P DS S trimer in the closed conformation at 2.9-Å resolution. b , Ribbon diagram of the SARS-CoV-2 2P DS S trimer atomic model in the same orientation as in panel a . In panels a and b , each S protomer is colored distinctly. Asterisks show the locations of the introduced disulfide bonds. c , Superimposition of the SARS-CoV-2 2P DS S trimer (green) to the coordinates from the 2.8-Å SARS-CoV-2 2P S structure in the closed conformation, PDB 6VXX (ref. 18 ) (black). d , Enlarged view of the designed disulfide bond with the corresponding region of cryo-EM density shown as a blue mesh.

    Techniques Used:

    Cryo-EM structure of the closed SARS-CoV-2 DS S glycoprotein. Zoomed-in view of the designed disulfide bond with the corresponding region of cryo-EM density shown as a blue mesh.
    Figure Legend Snippet: Cryo-EM structure of the closed SARS-CoV-2 DS S glycoprotein. Zoomed-in view of the designed disulfide bond with the corresponding region of cryo-EM density shown as a blue mesh.

    Techniques Used:

    Evaluation of SARS-CoV-2 2P DS S antigenicity. a – d , Binding to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black) of serially diluted concentrations of the human neutralizing antibodies S309 ( a ), S2H14 ( c ) and S304 ( d ) and the human ACE2 receptor fused to human Fc ( b ). e , Neutralization of SARS-CoV-2 S pseudovirus with human serum obtained from a patient with COVID-19. f , Binding of a serial dilution of the neutralizing convalescent serum shown in panel e to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black). Data are shown as mean and s.d. of n = 2 technical replicates; data are representative of two independent experiments. Data behind all graphs are available in Supplementary Data 1 . A 450 , absorbance at 450 nm; ND, not determined.
    Figure Legend Snippet: Evaluation of SARS-CoV-2 2P DS S antigenicity. a – d , Binding to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black) of serially diluted concentrations of the human neutralizing antibodies S309 ( a ), S2H14 ( c ) and S304 ( d ) and the human ACE2 receptor fused to human Fc ( b ). e , Neutralization of SARS-CoV-2 S pseudovirus with human serum obtained from a patient with COVID-19. f , Binding of a serial dilution of the neutralizing convalescent serum shown in panel e to immobilized SARS-CoV-2 2P DS S (green) or SARS-CoV-2 2P S (black). Data are shown as mean and s.d. of n = 2 technical replicates; data are representative of two independent experiments. Data behind all graphs are available in Supplementary Data 1 . A 450 , absorbance at 450 nm; ND, not determined.

    Techniques Used: Binding Assay, Neutralization, Serial Dilution

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Generated

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Standard Deviation

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

    Techniques Used: Serial Dilution, Standard Deviation, Generated

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.052233

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Generated

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

    Techniques Used: Generated

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Detection of anti-S1 IgG in recovered COVID-19 patients' serum. (A) Serial dilution tests with 10 representative samples, including six positive samples (PS), two negative samples (NS), and two commercially available negative controls (NC). Note that the positive/negative was determined with traditional plate-based ELISA. 200 X dilution in 2.5% BSA was determined to be the optimum dilution factor for differentiating the strong positive samples from the weak positive and negative samples. (B). Effective D006 concentrations for all nineteen samples and the two negative controls. The concentrations are marked as 0 ng/mL if the calculated concentration was below 2 ng/mL (too close to LLOD). The error bars are generated from duplicate measurements. Only four samples have effective D006 concentrations higher than 500 ng/mL after 200 times of dilution. (Note that PS4 and PS11 exceeded the upper limit of detection). (C). Statistical comparison between the negative samples and the positive samples. Since p
    Figure Legend Snippet: Detection of anti-S1 IgG in recovered COVID-19 patients' serum. (A) Serial dilution tests with 10 representative samples, including six positive samples (PS), two negative samples (NS), and two commercially available negative controls (NC). Note that the positive/negative was determined with traditional plate-based ELISA. 200 X dilution in 2.5% BSA was determined to be the optimum dilution factor for differentiating the strong positive samples from the weak positive and negative samples. (B). Effective D006 concentrations for all nineteen samples and the two negative controls. The concentrations are marked as 0 ng/mL if the calculated concentration was below 2 ng/mL (too close to LLOD). The error bars are generated from duplicate measurements. Only four samples have effective D006 concentrations higher than 500 ng/mL after 200 times of dilution. (Note that PS4 and PS11 exceeded the upper limit of detection). (C). Statistical comparison between the negative samples and the positive samples. Since p

    Techniques Used: Serial Dilution, Enzyme-linked Immunosorbent Assay, Concentration Assay, Generated

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

    Techniques Used: Serial Dilution, Standard Deviation, Generated

    34) Product Images from "SARS-CoV-2 induces activation and diversification of human plasmacytoid pre-dendritic cells"

    Article Title: SARS-CoV-2 induces activation and diversification of human plasmacytoid pre-dendritic cells

    Journal: bioRxiv

    doi: 10.1101/2020.07.10.197343

    SARS-CoV-2-induced pDC activation is inhibited by hydroxychloroquine. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI 1 with or without the presence of hydroxychloroquine (HCQ). (A) Dotplot showing pDC diversification in P1-, P2-, and P3-subpopulations in the presence of HCQ. (B) Quantification of the three populations. (C) Histograms of pDC’s activation markers. (D) Geometric mean (MFI) of activation markers. Histograms represent medians and bars interquartile of n=3 healthy donors. (E) Quantification of pro-inflammatory cytokines production. Bars represent medians of n=3 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2-induced pDC activation is inhibited by hydroxychloroquine. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI 1 with or without the presence of hydroxychloroquine (HCQ). (A) Dotplot showing pDC diversification in P1-, P2-, and P3-subpopulations in the presence of HCQ. (B) Quantification of the three populations. (C) Histograms of pDC’s activation markers. (D) Geometric mean (MFI) of activation markers. Histograms represent medians and bars interquartile of n=3 healthy donors. (E) Quantification of pro-inflammatory cytokines production. Bars represent medians of n=3 healthy donors. *P

    Techniques Used: Activation Assay, Cell Culture

    SARS-CoV-2-activated pDC produce pro-inflammatory cytokines. Sorted blood pDC from healthy donors were cultured for 24h or 48h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 1. (A) Quantification of pro-inflammatory cytokines at 24h. Bars represent medians of n=5 healthy donors. (B) Quantification of pro-inflammatory cytokines at 48h. Bars represent medians of n=3 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2-activated pDC produce pro-inflammatory cytokines. Sorted blood pDC from healthy donors were cultured for 24h or 48h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 1. (A) Quantification of pro-inflammatory cytokines at 24h. Bars represent medians of n=5 healthy donors. (B) Quantification of pro-inflammatory cytokines at 48h. Bars represent medians of n=3 healthy donors. *P

    Techniques Used: Cell Culture

    SARS-CoV-2 induces activation and diversification of primary human pDC. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, SARS-CoV-2, or Influenza virus A (Flu). (A) Dotplot showing pDC activation and diversification through the expression of PD-L1 and CD80 into P1-, P2-, and P3-subpopulations. (B) Quantification of the three populations. Bars represent medians of n=5 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2 induces activation and diversification of primary human pDC. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, SARS-CoV-2, or Influenza virus A (Flu). (A) Dotplot showing pDC activation and diversification through the expression of PD-L1 and CD80 into P1-, P2-, and P3-subpopulations. (B) Quantification of the three populations. Bars represent medians of n=5 healthy donors. *P

    Techniques Used: Activation Assay, Cell Culture, Expressing

    SARS-CoV-2 induces pDC activation in a dose dependent manner. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 0.04, 0.2, or 1. (A) Dotplot showing pDC activation through the expression of PD-L1 and CD80. (B) Quantification of the three populations. Bars represent medians of n=3 healthy donors. *P
    Figure Legend Snippet: SARS-CoV-2 induces pDC activation in a dose dependent manner. Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 0.04, 0.2, or 1. (A) Dotplot showing pDC activation through the expression of PD-L1 and CD80. (B) Quantification of the three populations. Bars represent medians of n=3 healthy donors. *P

    Techniques Used: Activation Assay, Cell Culture, Expressing

    35) Product Images from "SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques"

    Article Title: SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques

    Journal: bioRxiv

    doi: 10.1101/2020.07.07.191007

    Humoral responses to SARS-CoV-2 are dominated by IgG antibodies Concentrations of (A) IgM, (B) IgG, and (C) IgA antibodies specific for S1, S2, and N proteins were measured by BAMA or ELISA in serum of macaques infused with human COVID-19 convalescent plasma (CP; blue symbols) or naive plasma (NP; red symbols) and control non-infused animals (black symbols). The dashed line represents the median pre-infection (day 0) concentration for all animals. (D) The magnitude of the IgM, IgG and IgA antibody responses in animals that were not given human convalescent plasma was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Geometric mean fold increases with SEM are shown. (E) Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-RBD IgG antibodies measured by ELISA.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 are dominated by IgG antibodies Concentrations of (A) IgM, (B) IgG, and (C) IgA antibodies specific for S1, S2, and N proteins were measured by BAMA or ELISA in serum of macaques infused with human COVID-19 convalescent plasma (CP; blue symbols) or naive plasma (NP; red symbols) and control non-infused animals (black symbols). The dashed line represents the median pre-infection (day 0) concentration for all animals. (D) The magnitude of the IgM, IgG and IgA antibody responses in animals that were not given human convalescent plasma was determined by dividing post-infection concentrations by those measured on day 0 in each animal. Geometric mean fold increases with SEM are shown. (E) Correlations between day 10 levels of S1-specific IgG and IgM, N-specific IgA and IgG, and pseudovirus neutralizing antibody titers and anti-RBD IgG antibodies measured by ELISA.

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

    SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. (A) Gating strategy for identifying CD4 T cells in lung; red overlay represents paired CD4 subset from blood (either CD95- (naive) or CD95+ as indicated). (B) Scatter plot shows expression of Granzyme B, PD-1, CXCR3, CCR6 on CD69- and CD69+ subsets in lung and CD95+ CD4 T cells in blood. (C) Gating strategy for identification of GC T fh cells, GC B cells and FDCs. (D) Relative expression of Bcl-6, CD21, CD140b, and CXCR3 within GC cell subsets. (E) Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (*p
    Figure Legend Snippet: SARS-CoV-2 infection induces germinal center responses in mediastinal lymph nodes. (A) Gating strategy for identifying CD4 T cells in lung; red overlay represents paired CD4 subset from blood (either CD95- (naive) or CD95+ as indicated). (B) Scatter plot shows expression of Granzyme B, PD-1, CXCR3, CCR6 on CD69- and CD69+ subsets in lung and CD95+ CD4 T cells in blood. (C) Gating strategy for identification of GC T fh cells, GC B cells and FDCs. (D) Relative expression of Bcl-6, CD21, CD140b, and CXCR3 within GC cell subsets. (E) Frequency of GC T fh cells, GC B cells, FDCs significantly higher in mediastinal lymph node (*p

    Techniques Used: Infection, Expressing

    SARS-CoV-2 infection leads to a rapid and transient shift in innate immune responses and increases the number CD4 T follicular helper cells in peripheral blood. (A) Experimental design. Indian-origin rhesus macaques were inoculated with SARS-CoV-2 (SARS-CoV-2/human/USA/CA-CZB-59×002/2020) via the intranasal (IN), intratracheal (IT) and ocular route. Twenty-four hours later, animals were infused with either COVID-19 convalescent human plasma (I+CP; blue symbols), or normal plasma (I+NP; red symbols) (both at 4ml/kg), and four animals did not receive any plasma (infected; black symbols). Blood was sampled over the course of infection and tissues were collected at necropsy (11-14 DPI) for immune profiling. (B) Mean viral RNA (+range) in each of the groups within nasal washes (C) Flow plot illustrating gating strategy to identify innate immune subsets in whole blood. (D ) Kinetics of innate immune responses (*p
    Figure Legend Snippet: SARS-CoV-2 infection leads to a rapid and transient shift in innate immune responses and increases the number CD4 T follicular helper cells in peripheral blood. (A) Experimental design. Indian-origin rhesus macaques were inoculated with SARS-CoV-2 (SARS-CoV-2/human/USA/CA-CZB-59×002/2020) via the intranasal (IN), intratracheal (IT) and ocular route. Twenty-four hours later, animals were infused with either COVID-19 convalescent human plasma (I+CP; blue symbols), or normal plasma (I+NP; red symbols) (both at 4ml/kg), and four animals did not receive any plasma (infected; black symbols). Blood was sampled over the course of infection and tissues were collected at necropsy (11-14 DPI) for immune profiling. (B) Mean viral RNA (+range) in each of the groups within nasal washes (C) Flow plot illustrating gating strategy to identify innate immune subsets in whole blood. (D ) Kinetics of innate immune responses (*p

    Techniques Used: Infection

    CD4 T fh cells targeting the spike (S) and nucleocapsid (N) are generated following SARS-CoV-2 infection (A) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in spleen following stimulation with peptide megapools (B) Scatter plot showing AIM+ CD4 subsets; naive, CXCR5-, CXCR5+, and CXCR5+ PD-1 ++ GC T fh cells. The dashed line represents undetectable responses assigned a value of 0.01% ( C ) Cytokine profiles (IFN-γ, IL-2, TNFα, IL-17, IL-21) of CXCR5+, CXCR5-, and CD8+CD95+ T cells in spleen following PMA/Ionomycin stimulation. ( D ) Pie chart demonstrates polyfunctionality of T cell subsets following SARS-CoV-2 infection. (E ) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in PBMCs. (F) AIM+ CXCR5- and CXCR5+ CD4 subsets in PBMCs at Day 7. Black squares denote SARS-CoV-2 unexposed animals. Circles denote infected and triangles denote infected+infused animals.
    Figure Legend Snippet: CD4 T fh cells targeting the spike (S) and nucleocapsid (N) are generated following SARS-CoV-2 infection (A) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in spleen following stimulation with peptide megapools (B) Scatter plot showing AIM+ CD4 subsets; naive, CXCR5-, CXCR5+, and CXCR5+ PD-1 ++ GC T fh cells. The dashed line represents undetectable responses assigned a value of 0.01% ( C ) Cytokine profiles (IFN-γ, IL-2, TNFα, IL-17, IL-21) of CXCR5+, CXCR5-, and CD8+CD95+ T cells in spleen following PMA/Ionomycin stimulation. ( D ) Pie chart demonstrates polyfunctionality of T cell subsets following SARS-CoV-2 infection. (E ) Gating strategy for identifying SARS-CoV-2 specific CD4 T cells in PBMCs. (F) AIM+ CXCR5- and CXCR5+ CD4 subsets in PBMCs at Day 7. Black squares denote SARS-CoV-2 unexposed animals. Circles denote infected and triangles denote infected+infused animals.

    Techniques Used: Generated, Infection

    36) Product Images from "A human monoclonal antibody blocking SARS-CoV-2 infection"

    Article Title: A human monoclonal antibody blocking SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.987958

    Protein sequence alignment of the S1 B receptor binding in (RBD) of the SARS-CoV and SARS-CoV-2 spike proteins by ClustalW. Numbering denotes the residue position in the full-length spike protein of SARS-CoV (Genbank: AAP13441.1) and SARS-CoV-2 (Genbank: QHD43416.1). Asterisks (*) indicated fully conserved residues, the colon symbol (:) indicates conservation between groups of very similar properties, and the period symbol (.) indicates conservation between groups of weakly similar properties. Sequences corresponding to the S1 B receptor binding core domain and the receptor binding subdomain are colored in blue and orange, respectively. The fourteen residues that are involved in binding of SARS-CoV S1 B human ACE2 are highlighted in grey 1 .
    Figure Legend Snippet: Protein sequence alignment of the S1 B receptor binding in (RBD) of the SARS-CoV and SARS-CoV-2 spike proteins by ClustalW. Numbering denotes the residue position in the full-length spike protein of SARS-CoV (Genbank: AAP13441.1) and SARS-CoV-2 (Genbank: QHD43416.1). Asterisks (*) indicated fully conserved residues, the colon symbol (:) indicates conservation between groups of very similar properties, and the period symbol (.) indicates conservation between groups of weakly similar properties. Sequences corresponding to the S1 B receptor binding core domain and the receptor binding subdomain are colored in blue and orange, respectively. The fourteen residues that are involved in binding of SARS-CoV S1 B human ACE2 are highlighted in grey 1 .

    Techniques Used: Sequencing, Binding Assay

    The neutralizing 47D11 monoclonal antibody binds the receptor binding domain of SARS-CoV and SARS-CoV-2 spike proteins without eliminating S1 B /ACE2 receptor interaction. a) ELISA binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from at least two independent experiments performed is shown. b) Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analysed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Suppl.Fig.4 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analysed for (ACE2-)GFP expression (x-axis) and S1 B binding (y-axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. c) Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 18 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: Similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white.
    Figure Legend Snippet: The neutralizing 47D11 monoclonal antibody binds the receptor binding domain of SARS-CoV and SARS-CoV-2 spike proteins without eliminating S1 B /ACE2 receptor interaction. a) ELISA binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from at least two independent experiments performed is shown. b) Interference of antibodies with binding of the S-S1 B of SARS-CoV and SARS-CoV-2 to cell surface ACE2-GFP analysed by flow cytometry. Prior to cell binding, S1 B was mixed with mAb (mAbs 47D11, 35F4, 43C6, 7.7G6, in H2L2 format) with indicated specificity in a mAb:S1 B molar ratio of 8:1 (see Suppl.Fig.4 for an extensive analysis using different mAb:S1 B molar ratio’s). Cells are analysed for (ACE2-)GFP expression (x-axis) and S1 B binding (y-axis). Percentages of cells that scored negative, single positive, or double positive are shown in each quadrant. c) Divergence in surface residues in S1 B of SARS-CoV and SARS-CoV-2. Upper panel: Structure of the SARS-CoV spike protein S1 B RBD in complex with human ACE2 receptor (PDB: 2AJF) 18 . ACE2 (wheat color) is visualized in ribbon presentation. The S1 B core domain (blue) and subdomain (orange) are displayed in surface presentation using PyMOL, and are visualized with the same colors in the linear diagram of the spike protein above, with positions of the S1 and S2 subunits, the S ectodomain (S ecto ), the S1 domains S1 A-D and the transmembrane domain (TM) indicated. Lower panel: Similar as panel above with surface residues on S1 B of SARS-CoV that are at variance with SARS-CoV-2 colorored in white.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing

    H2L2 monoclonal antibodies 35F4 and 43C6 neutralize SARS-CoV but not SARS-CoV-2. Antibody-mediated neutralization of infection of VSV particles pseudotyped with spike proteins of SARS-CoV (upper panel) and SARS-CoV-2 (lower panel) by the 35F4 and 43C6 H2L2 antibodies targeting SARS-S1 but not SARS2-S1 (see Suppl.Fig.1 ). An irrelevant antibody was taken along as a human IgG1 isotype control. Means ± SD of triplicates are shown.
    Figure Legend Snippet: H2L2 monoclonal antibodies 35F4 and 43C6 neutralize SARS-CoV but not SARS-CoV-2. Antibody-mediated neutralization of infection of VSV particles pseudotyped with spike proteins of SARS-CoV (upper panel) and SARS-CoV-2 (lower panel) by the 35F4 and 43C6 H2L2 antibodies targeting SARS-S1 but not SARS2-S1 (see Suppl.Fig.1 ). An irrelevant antibody was taken along as a human IgG1 isotype control. Means ± SD of triplicates are shown.

    Techniques Used: Neutralization, Infection

    Binding kinetics of 47D11 to the S ectodomain and S1 B of SARS-CoV and SARS-CoV-2. Binding kinetics of 47D11 to immobilized recombinant SARS-S ecto , SARS2-S ecto , SARS-S1 B and SARS2-S1 B was measured using biolayer interferometry at 25°C, as described previously 21 . Kinetic binding assay was performed by loading 47D11 mAb at optimal concentration (42 nM) on anti-human Fc biosensor for 10 mins. Antigen association step was performed by incubating the sensor with a range of concentrations of the recombinant spike ectodomain (1600-800-400-200-100-50-25 nM) for 10 min, followed by a dissociation step in PBS for 60 min. The kinetics constants were calculated using 1:1 Langmuir binding model on Fortebio Data Analysis 7.0 software.
    Figure Legend Snippet: Binding kinetics of 47D11 to the S ectodomain and S1 B of SARS-CoV and SARS-CoV-2. Binding kinetics of 47D11 to immobilized recombinant SARS-S ecto , SARS2-S ecto , SARS-S1 B and SARS2-S1 B was measured using biolayer interferometry at 25°C, as described previously 21 . Kinetic binding assay was performed by loading 47D11 mAb at optimal concentration (42 nM) on anti-human Fc biosensor for 10 mins. Antigen association step was performed by incubating the sensor with a range of concentrations of the recombinant spike ectodomain (1600-800-400-200-100-50-25 nM) for 10 min, followed by a dissociation step in PBS for 60 min. The kinetics constants were calculated using 1:1 Langmuir binding model on Fortebio Data Analysis 7.0 software.

    Techniques Used: Binding Assay, Recombinant, Concentration Assay, Software

    47D11 neutralizes SARS-CoV and SARS-CoV-2. a) Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b) Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least two independent experiments performed is shown. Iso-CTRL: irrelevant isotype monoclonal antibody. c) Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown.
    Figure Legend Snippet: 47D11 neutralizes SARS-CoV and SARS-CoV-2. a) Binding of 47D11 to HEK-293T cells expressing GFP-tagged spike proteins of SARS-CoV and SARS-CoV-2 detected by immunofluorescence assay. The human mAb 7.7G6 targeting the MERS-CoV S1 B spike domain was taken along as a negative control, cell nuclei in the overlay images are visualized with DAPI. b) Antibody-mediated neutralization of infection of luciferase-encoding VSV particles pseudotyped with spike proteins of SARS-CoV and SARS-CoV-2. Pseudotyped VSV particles pre-incubated with antibodies at indicated concentrations (see methods) were used to infect VeroE6 cells and luciferase activities in cell lysates were determined at 24 h post transduction to calculate infection (%) relative to non-antibody-treated controls. The average ± SD from at least two independent experiments performed is shown. Iso-CTRL: irrelevant isotype monoclonal antibody. c) Antibody-mediated neutralization of SARS-CoV and SARS-CoV-2 infection on VeroE6 cells. The experiment was performed with triplicate samples, the average ± SD is shown.

    Techniques Used: Binding Assay, Expressing, Immunofluorescence, Negative Control, Neutralization, Infection, Luciferase, Incubation, Transduction

    37) Product Images from "Multivalency transforms SARS-CoV-2 antibodies into broad and ultrapotent neutralizers"

    Article Title: Multivalency transforms SARS-CoV-2 antibodies into broad and ultrapotent neutralizers

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.341636

    The Multabody enhances the potency of human mAbs from phage display. ( A ) Work flow for the identification of potent anti-SARS-CoV-2 neutralizers using the MB technology. ( B ) Comparison of neutralization potency between IgGs and MBs that display the same human Fab sequences derived from phage display. ( C ) IC 50 values fold increase upon multimerization. ( D ) Apparent affinity (K D ), association (k on ) and dissociation (k off ) rates of the most potent neutralizing MBs compared to their IgG counterparts for binding the SARS-CoV-2 S protein. Three biological replicates and their mean are shown for IC 50 values in (b) and (c).
    Figure Legend Snippet: The Multabody enhances the potency of human mAbs from phage display. ( A ) Work flow for the identification of potent anti-SARS-CoV-2 neutralizers using the MB technology. ( B ) Comparison of neutralization potency between IgGs and MBs that display the same human Fab sequences derived from phage display. ( C ) IC 50 values fold increase upon multimerization. ( D ) Apparent affinity (K D ), association (k on ) and dissociation (k off ) rates of the most potent neutralizing MBs compared to their IgG counterparts for binding the SARS-CoV-2 S protein. Three biological replicates and their mean are shown for IC 50 values in (b) and (c).

    Techniques Used: Neutralization, Derivative Assay, Binding Assay

    Binding profiles of IgGs and MBs. BLI response curves of IgG and MBs binding to RBD (left) and S protein (right) of SARS-CoV-2 immobilized onto Ni-NTA biosensors. 2-fold dilution series from 125 to 4 nM (IgG), and 16 to 0.5 nM (MB) were used. Black lines represent raw data whereas red lines represent global fits.
    Figure Legend Snippet: Binding profiles of IgGs and MBs. BLI response curves of IgG and MBs binding to RBD (left) and S protein (right) of SARS-CoV-2 immobilized onto Ni-NTA biosensors. 2-fold dilution series from 125 to 4 nM (IgG), and 16 to 0.5 nM (MB) were used. Black lines represent raw data whereas red lines represent global fits.

    Techniques Used: Binding Assay

    Epitope delineation of the most potent mAb specificities. ( A ) Surface and cartoon representation of RBD ( light green for the core and dark green for RBM) and ACE2 46 ( light brown ) binding. Heat map showing binding competition experiments. Epitope bins are highlighted by dashed-line boxes. ( B ) 15.0 Å filtered cryo-EM reconstruction of the Spike ( grey ) in complex with Fab 80 ( yellow ), 298 ( orange ) and 324 ( red ). The RBD and NTD are shown in green and blue , respectively. ( C ) Cryo-EM reconstruction of Fab 46 ( pink ) and RBD ( green ) complex. A RBD cartoon 46 is fitted into the partial density observed for the RBD. ( D ) Crystal structure of the ternary complex formed by Fab 52 ( purple ), Fab 298 ( orange ) and RBD ( green ). ( E ) Composite image depicting antibodies targeting SARS-CoV-2 S with available PDB or EMD entries 3 , 4 , 6 , 8 , 10 , 16 , 17 , 47 , 48 . Inset: close up view of antibodies targeting different antigenic sites on the RBD. The mAb with the lowest reported IC 50 value against SARS-CoV-2 PsV was selected as a representative antibody of the bin (color coding as in b).
    Figure Legend Snippet: Epitope delineation of the most potent mAb specificities. ( A ) Surface and cartoon representation of RBD ( light green for the core and dark green for RBM) and ACE2 46 ( light brown ) binding. Heat map showing binding competition experiments. Epitope bins are highlighted by dashed-line boxes. ( B ) 15.0 Å filtered cryo-EM reconstruction of the Spike ( grey ) in complex with Fab 80 ( yellow ), 298 ( orange ) and 324 ( red ). The RBD and NTD are shown in green and blue , respectively. ( C ) Cryo-EM reconstruction of Fab 46 ( pink ) and RBD ( green ) complex. A RBD cartoon 46 is fitted into the partial density observed for the RBD. ( D ) Crystal structure of the ternary complex formed by Fab 52 ( purple ), Fab 298 ( orange ) and RBD ( green ). ( E ) Composite image depicting antibodies targeting SARS-CoV-2 S with available PDB or EMD entries 3 , 4 , 6 , 8 , 10 , 16 , 17 , 47 , 48 . Inset: close up view of antibodies targeting different antigenic sites on the RBD. The mAb with the lowest reported IC 50 value against SARS-CoV-2 PsV was selected as a representative antibody of the bin (color coding as in b).

    Techniques Used: Binding Assay

    Neutralization and thermostability of SARS-CoV-2 RBD-targeting Multabodies and their parental IgGs. (A) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgGs (black) and MBs (dark red). The mean IC 50 value of three biological replicates, each with two technical replicates are displayed for comparison. ( B ) Comparison of the aggregation temperature (T agg ) of the seven most potent IgGs (white) and their respective MBs (dark red). ( C ) Static light scattering (SLS) at 266 nm versus temperature plots (dark red) from (b). T agg values are calculated from the maximum of the differential curves (light red) and indicated with yellow lines.
    Figure Legend Snippet: Neutralization and thermostability of SARS-CoV-2 RBD-targeting Multabodies and their parental IgGs. (A) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgGs (black) and MBs (dark red). The mean IC 50 value of three biological replicates, each with two technical replicates are displayed for comparison. ( B ) Comparison of the aggregation temperature (T agg ) of the seven most potent IgGs (white) and their respective MBs (dark red). ( C ) Static light scattering (SLS) at 266 nm versus temperature plots (dark red) from (b). T agg values are calculated from the maximum of the differential curves (light red) and indicated with yellow lines.

    Techniques Used: Neutralization, Titration

    Multabodies overcome SARS-CoV-2 sequence diversity. ( A ) Cartoon representation of the RBD showing four naturally occurring mutations as spheres. The epitopes of mAbs 52 ( light pink ) and 298 ( yellow ) are shown as representative epitopes of each bin. ( B ) Affinity and ( C ) IC 50 fold-change comparison between WT and mutated RBD and PsV, respectively. ( D ) Neutralization potency of IgG ( grey bars ) vs MB ( dark red bars ) against SARS-CoV-2 PsV variants in comparison to WT PsV. ( E ) Neutralization potency comparison of two IgG cocktails (three IgGs), monospecific MB cocktails (three MBs) and tri-specific MBs against WT SARS-CoV-2 PsV and variants. mAbs sensitive to one or more PsV variants were selected to generate the cocktails and the tri-specific MBs. The mean of three biological replicates are shown in (b), (c), (d) and (e).
    Figure Legend Snippet: Multabodies overcome SARS-CoV-2 sequence diversity. ( A ) Cartoon representation of the RBD showing four naturally occurring mutations as spheres. The epitopes of mAbs 52 ( light pink ) and 298 ( yellow ) are shown as representative epitopes of each bin. ( B ) Affinity and ( C ) IC 50 fold-change comparison between WT and mutated RBD and PsV, respectively. ( D ) Neutralization potency of IgG ( grey bars ) vs MB ( dark red bars ) against SARS-CoV-2 PsV variants in comparison to WT PsV. ( E ) Neutralization potency comparison of two IgG cocktails (three IgGs), monospecific MB cocktails (three MBs) and tri-specific MBs against WT SARS-CoV-2 PsV and variants. mAbs sensitive to one or more PsV variants were selected to generate the cocktails and the tri-specific MBs. The mean of three biological replicates are shown in (b), (c), (d) and (e).

    Techniques Used: Sequencing, Neutralization

    Protein engineering to multimerize IgG-like particles against SARS-CoV-2. ( A ) Schematic representation of the human apoferritin split design. ( B ) Negative stain electron micrograph of the MB. (Scale bar 50 nm). ( C ) Hydrodynamic radius (R h ) of the MB. ( D ) Avidity effect on the binding (apparent K D ) of A48 ( purple ) and BD23 ( gray ) to the SARS-CoV-2 Spike. ( E ) Kinetic curves of BD23 IgG and MB with different Fc sequence variants binding to FcγRI (top row), FcRn at endosomal pH (middle row) and FcRn at physiological pH (bottom row). Black lines represent raw data whereas red lines represent global fits. ( F ) Neutralization of SARS-CoV-2 PsV by A48 and BD23 IgGs and MBs. The mean values ± SD for two technical replicates for a representative experiment is shown.
    Figure Legend Snippet: Protein engineering to multimerize IgG-like particles against SARS-CoV-2. ( A ) Schematic representation of the human apoferritin split design. ( B ) Negative stain electron micrograph of the MB. (Scale bar 50 nm). ( C ) Hydrodynamic radius (R h ) of the MB. ( D ) Avidity effect on the binding (apparent K D ) of A48 ( purple ) and BD23 ( gray ) to the SARS-CoV-2 Spike. ( E ) Kinetic curves of BD23 IgG and MB with different Fc sequence variants binding to FcγRI (top row), FcRn at endosomal pH (middle row) and FcRn at physiological pH (bottom row). Black lines represent raw data whereas red lines represent global fits. ( F ) Neutralization of SARS-CoV-2 PsV by A48 and BD23 IgGs and MBs. The mean values ± SD for two technical replicates for a representative experiment is shown.

    Techniques Used: Staining, Binding Assay, Sequencing, Neutralization

    The MB platform potently overcomes SARS-CoV-2 sequence variability. (A) Comparison of the neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614G PsV (grey). ( B ) Schematic representation of a tri-specific MB generated by combination of three Fab specificities and the Fc fragment using the MB split design. ( C ) Cocktails and tri-specific MBs that combine the specificities of mAbs 298, 80 and 52, or 298, 324 and 46 were generated and tested against WT PsV. ( D ) Neutralization potency change of cocktails and tri-specific MBs against pseudotyped SARS-CoV-2 variants in comparison to WT PsV. PsV variants that were sensitive to individual antibodies within the cocktails were selected. The area within the dotted lines represent a 3-fold change in IC 50 value. This threshold was established as the cutoff to establish increased sensitivity (up bars) and increased resistance (down bars). ( E ) Neutralization titration curves showing three biological replicates of cocktails and tri-specific MBs against the authentic SARS-CoV-2/SB2-P4-PB strain 45 . Mean IC 50 values of three biological replicates are shown.
    Figure Legend Snippet: The MB platform potently overcomes SARS-CoV-2 sequence variability. (A) Comparison of the neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614G PsV (grey). ( B ) Schematic representation of a tri-specific MB generated by combination of three Fab specificities and the Fc fragment using the MB split design. ( C ) Cocktails and tri-specific MBs that combine the specificities of mAbs 298, 80 and 52, or 298, 324 and 46 were generated and tested against WT PsV. ( D ) Neutralization potency change of cocktails and tri-specific MBs against pseudotyped SARS-CoV-2 variants in comparison to WT PsV. PsV variants that were sensitive to individual antibodies within the cocktails were selected. The area within the dotted lines represent a 3-fold change in IC 50 value. This threshold was established as the cutoff to establish increased sensitivity (up bars) and increased resistance (down bars). ( E ) Neutralization titration curves showing three biological replicates of cocktails and tri-specific MBs against the authentic SARS-CoV-2/SB2-P4-PB strain 45 . Mean IC 50 values of three biological replicates are shown.

    Techniques Used: Sequencing, Neutralization, Generated, Titration

    Avidity enhances binding and neutralization of VHH against SARS-CoV-2. ( A ) Schematic representation of a monomeric VHH domain and its multimerization using a conventional Fc scaffold or human apoferritin. ( B ) Size exclusion chromatography and SDS-PAGE of apoferritin alone ( gray ) and VHH-72 apoferritin particles ( gold ). ( C ) Negative stain electron microscopy of VHH-72 apoferritin particles. (Scale bar 50 nm). ( D ) Comparison of the binding avidity (apparent K D ) of VHH-72 to SARS-CoV-2 S protein when displayed in a bivalent (red) or 24-mer (gold) format. Apparent K D higher than 10 −12 M (dash line) is beyond the instrument detection limit. ( E ) Neutralization potency against SARS-CoV-2 PsV (color coding is as in d). One representative out of two experiments with similar results is shown.
    Figure Legend Snippet: Avidity enhances binding and neutralization of VHH against SARS-CoV-2. ( A ) Schematic representation of a monomeric VHH domain and its multimerization using a conventional Fc scaffold or human apoferritin. ( B ) Size exclusion chromatography and SDS-PAGE of apoferritin alone ( gray ) and VHH-72 apoferritin particles ( gold ). ( C ) Negative stain electron microscopy of VHH-72 apoferritin particles. (Scale bar 50 nm). ( D ) Comparison of the binding avidity (apparent K D ) of VHH-72 to SARS-CoV-2 S protein when displayed in a bivalent (red) or 24-mer (gold) format. Apparent K D higher than 10 −12 M (dash line) is beyond the instrument detection limit. ( E ) Neutralization potency against SARS-CoV-2 PsV (color coding is as in d). One representative out of two experiments with similar results is shown.

    Techniques Used: Binding Assay, Neutralization, Size-exclusion Chromatography, SDS Page, Staining, Electron Microscopy

    Neutralization profiles of selected IgGs and MBs in different assays. ( A ) Similar neutralization profiles of IgGs (left) vs MBs (right) against pseudotyped SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (gray) target cells. The mean IC 50 value and individual IC 50 values of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. ( B ) Neutralization titration curves of three biological replicates (different shades of gray) against the authentic SARS-CoV-2/SB2-P4-PB strain 45 . The mean IC 50 is indicated. The less sensitive neutralization phenotype observed against authentic virus in comparison to PsV is in agreement with previous reports 5 , 13 , 14 , 17 . However, other studies have observed similar values 3 , 11 , 12 , 16 between the two assays. This discrepancy makes crosscomparison of antibody potencies against live replicating virus difficult and is likely due to differences in the length of time of the neutralization experiment. Short incubation times will minimize the number of replications that the virus can undergo, resembling the one replication cycle of the PsV assays.
    Figure Legend Snippet: Neutralization profiles of selected IgGs and MBs in different assays. ( A ) Similar neutralization profiles of IgGs (left) vs MBs (right) against pseudotyped SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (gray) target cells. The mean IC 50 value and individual IC 50 values of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. ( B ) Neutralization titration curves of three biological replicates (different shades of gray) against the authentic SARS-CoV-2/SB2-P4-PB strain 45 . The mean IC 50 is indicated. The less sensitive neutralization phenotype observed against authentic virus in comparison to PsV is in agreement with previous reports 5 , 13 , 14 , 17 . However, other studies have observed similar values 3 , 11 , 12 , 16 between the two assays. This discrepancy makes crosscomparison of antibody potencies against live replicating virus difficult and is likely due to differences in the length of time of the neutralization experiment. Short incubation times will minimize the number of replications that the virus can undergo, resembling the one replication cycle of the PsV assays.

    Techniques Used: Neutralization, Titration, Incubation

    38) Product Images from "Convergent Antibody Responses to SARS-CoV-2 in Convalescent Individuals"

    Article Title: Convergent Antibody Responses to SARS-CoV-2 in Convalescent Individuals

    Journal: Nature

    doi: 10.1038/s41586-020-2456-9

    Plasma antibodies against SARS-CoV-2. a-d, Graphs show results of ELISAs measuring plasma reactivity to RBD ( a, b ) and S protein ( c, d ). Left shows optical density units at 450 nm (OD, Y axis) and reciprocal plasma dilutions (X axis). Negative controls in black; individuals 21, and 47 in blue and red lines and arrowheads, respectively. Right shows normalized area under the curve (AUC) for 8 controls and each of 149 individuals in the cohort. e , Symptom (Sx) onset to time of sample collection in days (X axis) plotted against normalized AUC for IgM binding to RBD (Y axis); r=0.5517 and p=
    Figure Legend Snippet: Plasma antibodies against SARS-CoV-2. a-d, Graphs show results of ELISAs measuring plasma reactivity to RBD ( a, b ) and S protein ( c, d ). Left shows optical density units at 450 nm (OD, Y axis) and reciprocal plasma dilutions (X axis). Negative controls in black; individuals 21, and 47 in blue and red lines and arrowheads, respectively. Right shows normalized area under the curve (AUC) for 8 controls and each of 149 individuals in the cohort. e , Symptom (Sx) onset to time of sample collection in days (X axis) plotted against normalized AUC for IgM binding to RBD (Y axis); r=0.5517 and p=

    Techniques Used: Binding Assay

    Anti-SARS-CoV-2 RBD antibody reactivity. a, Graph shows results of ELISA assays measuring monoclonal antibody binding to RBD. Optical density units at 450 nm (OD, Y axis) vs. antibody concentrations (X axis); 94 samples and 1 isotype control. C121, C135 C144 and isotype control in red, green, purple, and black respectively, in all panels. b, Graph shows normalized relative luminescence values (RLU, Y axis) in cell lysates of 293T ACE2 cells 48 hours after infection with SARS-CoV-2 pseudovirus in the presence of increasing concentrations of monoclonal antibodies (X axis). 89 samples and 1 isotype control. c, Normalized RLU for SARS-CoV-2 pseudovirus neutralization (Y axis) vs. titration of monoclonal antibodies C121, C135 and C144. d, SARS-CoV-2 real virus neutralization assay. Normalized infected cells (Y axis, determined by dividing the amount of infection per well by the average of control wells infected in the absence of antibodies) vs. titration of monoclonal antibodies C121, C135 and C144. a to d show a representative of two independent experiments. In b and c is mean of duplicates and in d is mean with standard deviation of triplicates. e, IC 50 s for antibodies assayed in b and d , the average value of at least two experiments is shown. Samples with IC 50 s above 1μg/ml were plotted at 1μg/ml; n=89 (pseudovirus) and n=3 (virus), respectively. f, Diagrammatic representation of biolayer interferometry experiment. g, to RBD. h-n , Secondary antibody binding to preformed IgG-RBD complexes (Ab1). The table displays the shift in nanometers after second antibody (Ab2) binding to the antigen in the presence of the first antibody (Ab1). Values are normalized by the subtraction of the autologous antibody control. Representative of two experiments. o-q, Representative 2D-class averages and 3D reconstructed volumes for SARS-CoV-S 2P trimers complexed with C002, C119, and C121 Fabs. 2D class averages with observable Fab density are boxed. r , Overlay of S-Fab complexes with fully-occupied C002 (blue), C121 (magenta) and C119 (orange) Fabs. The SARS-CoV-2 S model from PDB 6VYB was fit into the density and the SARS-CoV mAb S230 (PDB 6NB6) is shown as a reference (green ribbon).
    Figure Legend Snippet: Anti-SARS-CoV-2 RBD antibody reactivity. a, Graph shows results of ELISA assays measuring monoclonal antibody binding to RBD. Optical density units at 450 nm (OD, Y axis) vs. antibody concentrations (X axis); 94 samples and 1 isotype control. C121, C135 C144 and isotype control in red, green, purple, and black respectively, in all panels. b, Graph shows normalized relative luminescence values (RLU, Y axis) in cell lysates of 293T ACE2 cells 48 hours after infection with SARS-CoV-2 pseudovirus in the presence of increasing concentrations of monoclonal antibodies (X axis). 89 samples and 1 isotype control. c, Normalized RLU for SARS-CoV-2 pseudovirus neutralization (Y axis) vs. titration of monoclonal antibodies C121, C135 and C144. d, SARS-CoV-2 real virus neutralization assay. Normalized infected cells (Y axis, determined by dividing the amount of infection per well by the average of control wells infected in the absence of antibodies) vs. titration of monoclonal antibodies C121, C135 and C144. a to d show a representative of two independent experiments. In b and c is mean of duplicates and in d is mean with standard deviation of triplicates. e, IC 50 s for antibodies assayed in b and d , the average value of at least two experiments is shown. Samples with IC 50 s above 1μg/ml were plotted at 1μg/ml; n=89 (pseudovirus) and n=3 (virus), respectively. f, Diagrammatic representation of biolayer interferometry experiment. g, to RBD. h-n , Secondary antibody binding to preformed IgG-RBD complexes (Ab1). The table displays the shift in nanometers after second antibody (Ab2) binding to the antigen in the presence of the first antibody (Ab1). Values are normalized by the subtraction of the autologous antibody control. Representative of two experiments. o-q, Representative 2D-class averages and 3D reconstructed volumes for SARS-CoV-S 2P trimers complexed with C002, C119, and C121 Fabs. 2D class averages with observable Fab density are boxed. r , Overlay of S-Fab complexes with fully-occupied C002 (blue), C121 (magenta) and C119 (orange) Fabs. The SARS-CoV-2 S model from PDB 6VYB was fit into the density and the SARS-CoV mAb S230 (PDB 6NB6) is shown as a reference (green ribbon).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Infection, Neutralization, Titration, Standard Deviation

    Binding of the monoclonal antibodies to the RBD of SARS-CoV-2 and cross-reactivity to SARS-CoV. a, EC 50 values for binding to the RBD of SARS-CoV-2. Average of two or more experiments; n=89. b and c, Binding curves and EC 50 values (average of two experiments) for binding to the RBD of SARS-CoV; n=20 and n=17 (excluding isotype and CR3022), respectively. d and e , SARS-CoV pseudovirus neutralization curves and IC 50 values. Shown in d are the standard deviations of duplicates for one representative experiment and in e is the average of two experiments (n=10, excluding CR3022). Samples with IC 50 s above 1μg/ml were plotted at 1μg/ml.
    Figure Legend Snippet: Binding of the monoclonal antibodies to the RBD of SARS-CoV-2 and cross-reactivity to SARS-CoV. a, EC 50 values for binding to the RBD of SARS-CoV-2. Average of two or more experiments; n=89. b and c, Binding curves and EC 50 values (average of two experiments) for binding to the RBD of SARS-CoV; n=20 and n=17 (excluding isotype and CR3022), respectively. d and e , SARS-CoV pseudovirus neutralization curves and IC 50 values. Shown in d are the standard deviations of duplicates for one representative experiment and in e is the average of two experiments (n=10, excluding CR3022). Samples with IC 50 s above 1μg/ml were plotted at 1μg/ml.

    Techniques Used: Binding Assay, Neutralization

    Neutralization of SARS-CoV-2 pseudovirus by plasma. a, Graph shows normalized relative luminescence values (RLU, Y axis) in cell lysates of 293T ACE2 cells 48 hours after infection with nanoluc-expressing SARS-CoV-2 pseudovirus in the presence of increasing concentrations of plasma (X axis) derived from 149 participants (grey, except individuals 21 and 47 in blue and red lines, bars and arrowheads, respectively) and 3 negative controls (black lines). Mean of duplicates; representative of two independent experiments. b, Ranked average half-maximal inhibitory plasma neutralizing titer (NT 50 ) for the 59 of 149 individuals with NT 50 s > 500 and individual 107. Asterisks indicate donors from which antibody sequences were derived. c, Normalized AUC for anti-RBD IgG ELISA (X axis) plotted against NT 50 (Y axis); r=0.6432, p=
    Figure Legend Snippet: Neutralization of SARS-CoV-2 pseudovirus by plasma. a, Graph shows normalized relative luminescence values (RLU, Y axis) in cell lysates of 293T ACE2 cells 48 hours after infection with nanoluc-expressing SARS-CoV-2 pseudovirus in the presence of increasing concentrations of plasma (X axis) derived from 149 participants (grey, except individuals 21 and 47 in blue and red lines, bars and arrowheads, respectively) and 3 negative controls (black lines). Mean of duplicates; representative of two independent experiments. b, Ranked average half-maximal inhibitory plasma neutralizing titer (NT 50 ) for the 59 of 149 individuals with NT 50 s > 500 and individual 107. Asterisks indicate donors from which antibody sequences were derived. c, Normalized AUC for anti-RBD IgG ELISA (X axis) plotted against NT 50 (Y axis); r=0.6432, p=

    Techniques Used: Neutralization, Infection, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Diagrammatic representation of the SARS-CoV-2 pseudovirus luciferase assay. a, Co-transfection of pNL4-3ΔEnv-nanoluc and pSARS-CoV-2 spike vectors into 293T cells (ATCC) leads to production of SARS-CoV-2 Spike-pseudotyped HIV-1 particles (SARS-CoV-2 pseudovirus) carrying the Nanoluc gene. b, SARS-CoV-2 pseudovirus is incubated for 1 h at 37°C with plasma or monoclonal antibody dilutions. The virus-antibody mixture is used to infect ACE2-expressing 293T cells, which will express nanoluc Luciferase upon infection. c, Relative luminescence units (RLU) reads from lysates of ACE2-expressing 293T cells infected with increasing amounts of SARS-CoV-2 pseudovirus. Error bars represent standard deviation of triplicates, two experiments.
    Figure Legend Snippet: Diagrammatic representation of the SARS-CoV-2 pseudovirus luciferase assay. a, Co-transfection of pNL4-3ΔEnv-nanoluc and pSARS-CoV-2 spike vectors into 293T cells (ATCC) leads to production of SARS-CoV-2 Spike-pseudotyped HIV-1 particles (SARS-CoV-2 pseudovirus) carrying the Nanoluc gene. b, SARS-CoV-2 pseudovirus is incubated for 1 h at 37°C with plasma or monoclonal antibody dilutions. The virus-antibody mixture is used to infect ACE2-expressing 293T cells, which will express nanoluc Luciferase upon infection. c, Relative luminescence units (RLU) reads from lysates of ACE2-expressing 293T cells infected with increasing amounts of SARS-CoV-2 pseudovirus. Error bars represent standard deviation of triplicates, two experiments.

    Techniques Used: Luciferase, Cotransfection, Incubation, Expressing, Infection, Standard Deviation

    39) 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

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

    Techniques Used: Serial Dilution, Standard Deviation, Generated

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

    Techniques Used: Standard Deviation

    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

    Related Articles

    Clone Assay:

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: .. To evaluate the differences in antibody’s affinity towards SARS-CoV-2 S1 and SARS-CoV S1, we performed a side-by-side study with these two types of S1 proteins for all three clones of antibodies. .. In general, the chemiluminescent intensities are proportional to the concentration of the spiked-in monoclonal antibodies.

    Binding Assay:

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: .. For D001 and D006, the signal for both types of S1 proteins is very similar, indicating that the antibodies’ binding affinity towards SARS-CoV-2 S1 and SARS-CoV S1 should be very similar (D006 may have a slightly higher affinity towards SARS-CoV-2 S1 than SARS-CoV S1). .. However, for CR3022, the signal for SARS-CoV-2 S1 is systematically lower than that for SARS-CoV S1, indicative of a weaker affinity of CR3022 towards SARS-CoV-2 S1 than SARS-CoV S1, which agrees with recently published findings about CR3022’s binding ability , .

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: .. For example, CR3022’s binding affinity towards SARS-CoV S1 is stronger than its affinity towards SARS-CoV-2 S1. .. Conversely, D006’s binding affinity towards SARS-CoV S1 is weaker than SARS-CoV-2 S1.

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: .. However, for CR3022, the signal for SARS-CoV-2 S1 is systematically lower than that for SARS-CoV S1, indicative of a weaker affinity of CR3022 towards SARS-CoV-2 S1 than SARS-CoV S1, which agrees with recently published findings about CR3022’s binding ability , . ..

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: .. Conversely, D006’s binding affinity towards SARS-CoV S1 is weaker than SARS-CoV-2 S1. .. In addition, the pattern of calibration curves with SARS-CoV-2 S1 is significantly different from that with SARS-CoV S1.

    other:

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: While D001, D006, and CR3022’s affinities towards SARS-CoV-2 S1 vary significantly, they appear to be very similar to each other towards SARS-CoV S1.

    Article Title: Rapid and quantitative detection of COVID-19 markers in micro-liter sized samples
    Article Snippet: In contrast, as shown in the antibodies’ affinity towards SARS-CoV S1 can be very different from SARS-CoV-2 S1.

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    Screening <t>SARS-CoV-2</t> antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p
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    Screening SARS-CoV-2 antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p

    Journal: bioRxiv

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    doi: 10.1101/2020.12.30.424862

    Figure Lengend Snippet: Screening SARS-CoV-2 antiviral activity using the FDA-approved and bioactive compound libraries. (A) Assay scheme: Cells are treated with DMSO (left panel) or drug (middle and right panels), infected with SARS-CoV-2 or left uninfected (right panel) and incubated for 72-96h to observe cytopathic effect (CPE). CPE is measured by CTG assay, quantifying ATP content in viable cells using luminescence (RLU). The right panel shows the cytotoxicity control, treating cells with drugs but without virus. (B-C) Average luminescence is shown for (B) Vero-E6 at 72h or (C) Calu-3 cells at 96h post-infection. (D) Screen of FDA-approved and bioactive compound libraries on Vero-E6 cells with inhibition of CPE (%) on the y-axis and cell viability (%) on the x-axis normalized to DMSO-treated wells. Red: high priority hits with a cutoff of > 20% inhibition of CPE and > 70% cell viability. (E) As in (D), but on Calu-3 cells, with a cutoff of > 70% inhibition of CPE and > 70% cell viability. (F) Combination of inhibition of CPE (%) on Vero-E6 (y-axis) from (D) and Calu-3 (x-axis) from (E). (G) Gene set enrichment analysis. Distribution of the enrichment score (green line) across compounds annotated to molecular targets (vertical black lines). CDK1, CDK2, GSK-3 p

    Article Snippet: Immunofluorescence microscopy analysis (IFA) 1×104 Vero-E6, HPMEC/hACE2, or Huh-7 cells were seeded in black 96-well plates with clear bottoms 24 hours before adding drug combinations and infecting with SARS-CoV-2 at MOI 0.05 (viral inoculums were not washed away).

    Techniques: Activity Assay, Infection, Incubation, CTG Assay, Inhibition

    (A) HPMEC, (B) BEAS-2B, (C) HCT-116, (D) LNCaP, (E) HaCaT, (F) RD, (G) NCI-H1437, (H) Huh-7.5.1, (I) Caco-2, (J) A549/hACE2, (K) HBEC-30KT, or (L) A549 cells were infected with SARS-CoV-2 at MOI 0.5 or 0.05 as in figure 1 . Viral titers were analyzed by TCID50 assay at the indicated time points (hours post-infection, hpi). Dashed lines represent limit of detection. Data represent mean ± SEM for n = 2 independent experiments.

    Journal: bioRxiv

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    doi: 10.1101/2020.12.30.424862

    Figure Lengend Snippet: (A) HPMEC, (B) BEAS-2B, (C) HCT-116, (D) LNCaP, (E) HaCaT, (F) RD, (G) NCI-H1437, (H) Huh-7.5.1, (I) Caco-2, (J) A549/hACE2, (K) HBEC-30KT, or (L) A549 cells were infected with SARS-CoV-2 at MOI 0.5 or 0.05 as in figure 1 . Viral titers were analyzed by TCID50 assay at the indicated time points (hours post-infection, hpi). Dashed lines represent limit of detection. Data represent mean ± SEM for n = 2 independent experiments.

    Article Snippet: Immunofluorescence microscopy analysis (IFA) 1×104 Vero-E6, HPMEC/hACE2, or Huh-7 cells were seeded in black 96-well plates with clear bottoms 24 hours before adding drug combinations and infecting with SARS-CoV-2 at MOI 0.05 (viral inoculums were not washed away).

    Techniques: Infection, TCID50 Assay

    Dose response curves of compounds with SARS-CoV-2 antiviral activity. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Journal: bioRxiv

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    doi: 10.1101/2020.12.30.424862

    Figure Lengend Snippet: Dose response curves of compounds with SARS-CoV-2 antiviral activity. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Article Snippet: Immunofluorescence microscopy analysis (IFA) 1×104 Vero-E6, HPMEC/hACE2, or Huh-7 cells were seeded in black 96-well plates with clear bottoms 24 hours before adding drug combinations and infecting with SARS-CoV-2 at MOI 0.05 (viral inoculums were not washed away).

    Techniques: Activity Assay, Infection, Inhibition

    Confirmation and characterization of SARS-CoV-2 antiviral candidate compounds. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05, treated with the top 12 compounds (shown in Figure 3 ), disulfiram, or apilimod mesylate at indicated concentrations and supernatants were collected at 24 hpi. Viral titers and genome copies were calculated by TCID50 and qRT-PCR, respectively. (A) and (B) protein kinase and protease inhibitors, (C) and (D) anti-inflammatory compounds, (E) and (F) direct-acting antivirals and (G) and (H) other host-targeting compounds. TCID50 data represent mean ± SD for n = 2 independent experiments. Genome copy data represent mean ± SEM for n = 2 technical replicates and are representative of n = 2 independent experiments.

    Journal: bioRxiv

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    doi: 10.1101/2020.12.30.424862

    Figure Lengend Snippet: Confirmation and characterization of SARS-CoV-2 antiviral candidate compounds. Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05, treated with the top 12 compounds (shown in Figure 3 ), disulfiram, or apilimod mesylate at indicated concentrations and supernatants were collected at 24 hpi. Viral titers and genome copies were calculated by TCID50 and qRT-PCR, respectively. (A) and (B) protein kinase and protease inhibitors, (C) and (D) anti-inflammatory compounds, (E) and (F) direct-acting antivirals and (G) and (H) other host-targeting compounds. TCID50 data represent mean ± SD for n = 2 independent experiments. Genome copy data represent mean ± SEM for n = 2 technical replicates and are representative of n = 2 independent experiments.

    Article Snippet: Immunofluorescence microscopy analysis (IFA) 1×104 Vero-E6, HPMEC/hACE2, or Huh-7 cells were seeded in black 96-well plates with clear bottoms 24 hours before adding drug combinations and infecting with SARS-CoV-2 at MOI 0.05 (viral inoculums were not washed away).

    Techniques: Infection, Quantitative RT-PCR

    Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Journal: bioRxiv

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    doi: 10.1101/2020.12.30.424862

    Figure Lengend Snippet: Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with compounds at indicated concentrations. Data show % CPE inhibition in SARS-CoV-2 infected cells (red) and % cell viability in uninfected cells (black). Data are normalized to the mean of DMSO-treated wells and represent mean ± SD for n = 2 technical replicates.

    Article Snippet: Immunofluorescence microscopy analysis (IFA) 1×104 Vero-E6, HPMEC/hACE2, or Huh-7 cells were seeded in black 96-well plates with clear bottoms 24 hours before adding drug combinations and infecting with SARS-CoV-2 at MOI 0.05 (viral inoculums were not washed away).

    Techniques: Infection, Inhibition

    B02 synergy with remdesivir. (A) Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with 2 μM remdesivir, 10 μM B02, or a combination of 2 μM remdesivir and 10 μM B02 for 72h. CPE inhibition was measured by CTG assay. (B) Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with remdesivir at indicated concentrations in the presence or absence of 10 μM B02 for 96h. CPE inhibition was measured by CTG assay and was normalized to DMSO-treated wells. Data represent mean ± SD for n = 2 technical replicates.

    Journal: bioRxiv

    Article Title: Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

    doi: 10.1101/2020.12.30.424862

    Figure Lengend Snippet: B02 synergy with remdesivir. (A) Vero-E6 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with 2 μM remdesivir, 10 μM B02, or a combination of 2 μM remdesivir and 10 μM B02 for 72h. CPE inhibition was measured by CTG assay. (B) Calu-3 cells were infected with SARS-CoV-2 at MOI 0.05 and treated with remdesivir at indicated concentrations in the presence or absence of 10 μM B02 for 96h. CPE inhibition was measured by CTG assay and was normalized to DMSO-treated wells. Data represent mean ± SD for n = 2 technical replicates.

    Article Snippet: Immunofluorescence microscopy analysis (IFA) 1×104 Vero-E6, HPMEC/hACE2, or Huh-7 cells were seeded in black 96-well plates with clear bottoms 24 hours before adding drug combinations and infecting with SARS-CoV-2 at MOI 0.05 (viral inoculums were not washed away).

    Techniques: Infection, Inhibition, CTG Assay

    Spike mediated particle entry ( A ) Generation of pseudotyped lentiviral vectors. Second generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor binding domain (RBD, orange) are indicated. ( B ) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s, bottom blot for 5 s. ( C ) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μl vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Grey shaded area indicates 95% CI of signals from untransduced cells (blanks). ( D ) Effect of ACE2-overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95% CIs.

    Journal: bioRxiv

    Article Title: Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

    doi: 10.1101/2020.10.15.340604

    Figure Lengend Snippet: Spike mediated particle entry ( A ) Generation of pseudotyped lentiviral vectors. Second generation LVs pseudotyped with S protein were generated by transfection of HEK-293T cells with a packaging plasmid encoding HIV-1 gag/pol, a transfer vector plasmid with a lacZ reporter gene and one of two envelope plasmids encoding codon-optimized SARS-CoV-2 S with or without (SΔ19) the 19 C-terminal amino acids. The C-terminal endoplasmic reticulum retention signal (purple) and the receptor binding domain (RBD, orange) are indicated. ( B ) Incorporation of S protein into LVs determined by Western blotting. S-LV and SΔ19-LV particles (V) and lysates of their producer cells (C) were stained for the presence of S protein (top) and p24 as particle loading control. Top blot was exposed for 30 s, bottom blot for 5 s. ( C ) Gene transfer activities on the indicated cell lines. The indicated dilutions of 5 μl vector stock of SΔ19-LV or VSV-LV were added to the cells. Cell lysates were prepared three days after vector addition and lacZ reporter activity was quantified as a luminescence readout. Symbols represent means of technical triplicates. Grey shaded area indicates 95% CI of signals from untransduced cells (blanks). ( D ) Effect of ACE2-overexpression on reporter transfer. 293T cells transfected with ACE2 expression plasmid or mock plasmid were incubated with 0.2 μL of SΔ19-LV or VSV-LV. Cell lysates were prepared three days after vector addition and reporter activity was quantified as a luminescence readout. Bars represent geometric means of technical triplicates ±95% CIs.

    Article Snippet: Sera were from two convalescent patients who had been diagnosed with SARS-CoV-2 by PCR from throat swabs approximately 4 months prior to donation.

    Techniques: Generated, Transfection, Plasmid Preparation, Binding Assay, Western Blot, Staining, Activity Assay, Over Expression, Expressing, Incubation

    Family of symptomatic SARS-CoV-2 PCR positive parents and SARS-CoV-2 PCR negative children have distinct serological responses compared to healthy individuals, characterized by elevated SARS-CoV-2 specific responses. a PLSDA scores plot of healthy (blue triangles) vs family (circles) containing both SARS-CoV-2 PCR positive parents (orange) and negative children (yellow) exhibited 98.0% calibration and 96.0% cross-validation accuracy, with 62.7% of variance explained by LV1 (x-axis). Family member samples are labeled with A (adult) or C (child) with the day of sample collection listed after D. b PLSDA plot of LV1 loadings driving the separation of groups, where negatively loaded features are associated with the family members. c Hierarchical clustering of healthy individuals (blue) and family members (parents, orange; children, yellow) using a feature-selected serological signature, where red indicates a relatively high antibody response and blue a relatively low antibody response (z-score). Samples (x-axis) are labeled with H (healthy non-household members), and A (adult) or C (child). Day of sample collection is listed at the end of family member sample labels.

    Journal: Nature Communications

    Article Title: Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19

    doi: 10.1038/s41467-020-19545-8

    Figure Lengend Snippet: Family of symptomatic SARS-CoV-2 PCR positive parents and SARS-CoV-2 PCR negative children have distinct serological responses compared to healthy individuals, characterized by elevated SARS-CoV-2 specific responses. a PLSDA scores plot of healthy (blue triangles) vs family (circles) containing both SARS-CoV-2 PCR positive parents (orange) and negative children (yellow) exhibited 98.0% calibration and 96.0% cross-validation accuracy, with 62.7% of variance explained by LV1 (x-axis). Family member samples are labeled with A (adult) or C (child) with the day of sample collection listed after D. b PLSDA plot of LV1 loadings driving the separation of groups, where negatively loaded features are associated with the family members. c Hierarchical clustering of healthy individuals (blue) and family members (parents, orange; children, yellow) using a feature-selected serological signature, where red indicates a relatively high antibody response and blue a relatively low antibody response (z-score). Samples (x-axis) are labeled with H (healthy non-household members), and A (adult) or C (child). Day of sample collection is listed at the end of family member sample labels.

    Article Snippet: Saliva from a convalescent individual recently infected with SARS-CoV-2 was used as a positive control.

    Techniques: Polymerase Chain Reaction, Labeling

    Salivary and plasma antibody responses against SARS-CoV-2 S1 protein by ELISA and by microneutralization assay. a Anti-S1 salivary IgA, IgG, and IgM. # IgA anti-S1 response that developed concurrent with resolution of symptoms. b Anti-S1 plasma IgA, IgG, and IgM. c Neutralizing antibody activity in plasma. A1: mother, A2: father, C1: male (9 years), C2: male (7 years), C3: female (5 years), (P) positive control.

    Journal: Nature Communications

    Article Title: Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19

    doi: 10.1038/s41467-020-19545-8

    Figure Lengend Snippet: Salivary and plasma antibody responses against SARS-CoV-2 S1 protein by ELISA and by microneutralization assay. a Anti-S1 salivary IgA, IgG, and IgM. # IgA anti-S1 response that developed concurrent with resolution of symptoms. b Anti-S1 plasma IgA, IgG, and IgM. c Neutralizing antibody activity in plasma. A1: mother, A2: father, C1: male (9 years), C2: male (7 years), C3: female (5 years), (P) positive control.

    Article Snippet: Saliva from a convalescent individual recently infected with SARS-CoV-2 was used as a positive control.

    Techniques: Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Activity Assay, Positive Control

    Antibody responses in patients with COVID-19. ( A ) Levels of serum IgG, IgA, and IgM antibodies against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 were measured by ELISA. Blue (IgG), green (IgA), and purple (IgM) lines represent the antibody titers of individual COVID-19 positive sera, pink lines represent negative control sera. ( B ) Each dot represents an individual antibody titer, and groups are divided by age and sex. ( C ) Correlation of anti-N responses with other antiviral protein responses in patients with COVID-19. Correlations of the values were assessed by linear regression. ( D ) Correlations between neutralization titer (x-axis) and each antibody response to the viral protein of SARS-CoV-2 (y-axis) are shown. Each dot indicates the neutralization titer of serum antibodies of patients with COVID-19, as determined by calculating the highest dilution of serum that prevents infection of 50% of inoculations and antiviral protein titers (IgG) of human sera, as determined by ELISA.

    Journal: Vaccines

    Article Title: Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

    doi: 10.3390/vaccines8040684

    Figure Lengend Snippet: Antibody responses in patients with COVID-19. ( A ) Levels of serum IgG, IgA, and IgM antibodies against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2 were measured by ELISA. Blue (IgG), green (IgA), and purple (IgM) lines represent the antibody titers of individual COVID-19 positive sera, pink lines represent negative control sera. ( B ) Each dot represents an individual antibody titer, and groups are divided by age and sex. ( C ) Correlation of anti-N responses with other antiviral protein responses in patients with COVID-19. Correlations of the values were assessed by linear regression. ( D ) Correlations between neutralization titer (x-axis) and each antibody response to the viral protein of SARS-CoV-2 (y-axis) are shown. Each dot indicates the neutralization titer of serum antibodies of patients with COVID-19, as determined by calculating the highest dilution of serum that prevents infection of 50% of inoculations and antiviral protein titers (IgG) of human sera, as determined by ELISA.

    Article Snippet: Protein Microarray Assays Antigens printed onto microarrays were glycoproteins or nucleoproteins of HCoVs, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, respiratory syncytial viruses (RSVs), metapneumoviruses (MPVs), parainfluenza viruses (PIVs), adenoviruses (AdVs), and influenza viruses.

    Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Neutralization, Infection

    Immune responses of mice to SARS-CoV-2. ( A ) Viral protein-specific IgG antibody responses of mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2. Viral protein-specific IgG responses of murine sera and cross-reactivities to the viral proteins of SARS-CoV, CCoV 1-71, HCoV NL63, and influenza virus were measured by ELISA. Blue bars represent homologous antibody responses, and bars with slash lines display cross-reactive antibody responses. Red lines are the cut-offs calculated as 3 times the mean absorbance value of the preimmune sera. ( B ) Antibody response was measured in mice immunized with S, N, E, M, or 3CLpro of SARS-CoV. Purple bars show homologous antibody responses, and bars with slash lines represent cross-reactive antibody responses. ( C ) S-, S1-, RBD-, S2-, N-, E-, and NS3-specific IgG PCs in spinal bone marrow (left) and ASCs in cervical lymph nodes (right) were quantified ex vivo in ELISpot assays. Bone marrow and lymph node cells were obtained from the mice immunized with respective viral protein of SARS-CoV-2. ( D ) The presence of neutralizing antibodies in mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2; inactivated whole virus of SARS-CoV-2; and S, N, E, M, or 3CLpro of SARS-CoV was measured by in vitro neutralization assays using a pseudotyped virus. The transduction efficacy was determined by measuring the luciferase activity. Nonlinear regression analysis was used to obtain the IC 50 in the neutralization assays.

    Journal: Vaccines

    Article Title: Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

    doi: 10.3390/vaccines8040684

    Figure Lengend Snippet: Immune responses of mice to SARS-CoV-2. ( A ) Viral protein-specific IgG antibody responses of mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2. Viral protein-specific IgG responses of murine sera and cross-reactivities to the viral proteins of SARS-CoV, CCoV 1-71, HCoV NL63, and influenza virus were measured by ELISA. Blue bars represent homologous antibody responses, and bars with slash lines display cross-reactive antibody responses. Red lines are the cut-offs calculated as 3 times the mean absorbance value of the preimmune sera. ( B ) Antibody response was measured in mice immunized with S, N, E, M, or 3CLpro of SARS-CoV. Purple bars show homologous antibody responses, and bars with slash lines represent cross-reactive antibody responses. ( C ) S-, S1-, RBD-, S2-, N-, E-, and NS3-specific IgG PCs in spinal bone marrow (left) and ASCs in cervical lymph nodes (right) were quantified ex vivo in ELISpot assays. Bone marrow and lymph node cells were obtained from the mice immunized with respective viral protein of SARS-CoV-2. ( D ) The presence of neutralizing antibodies in mice immunized with S, S1, RBD, S2, N, E, or NS3 of SARS-CoV-2; inactivated whole virus of SARS-CoV-2; and S, N, E, M, or 3CLpro of SARS-CoV was measured by in vitro neutralization assays using a pseudotyped virus. The transduction efficacy was determined by measuring the luciferase activity. Nonlinear regression analysis was used to obtain the IC 50 in the neutralization assays.

    Article Snippet: Protein Microarray Assays Antigens printed onto microarrays were glycoproteins or nucleoproteins of HCoVs, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, respiratory syncytial viruses (RSVs), metapneumoviruses (MPVs), parainfluenza viruses (PIVs), adenoviruses (AdVs), and influenza viruses.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Enzyme-linked Immunospot, In Vitro, Neutralization, Transduction, Luciferase, Activity Assay

    Antibody responses in dogs and cats. ( A ) Serum antibody responses of dogs to S, S1, RBD, S2, N, E, and NS3 viral proteins of SARS-CoV-2, and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by IgG ELISA. The OD values were normalized by subtracting the OD values generated by sera from SPF dogs. ( B ) In canine serum samples, IgG levels against CCoV/HCoV and those against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2, as detected by ELISA, were used in correlation analyses. ( C ) Serum IgG antibody responses of cats to SARS-CoV-2 S, S1, RBD, S2, N, E, and NS3, and and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by ELISA. The crude OD values generated by ELISA were normalized by subtracting the OD values generated by sera from SPF cats. ( D ) ELISA was also used to determine IgG antibody levels against N of SARS-CoV-2, CCoV 1-71, and HCoV NL63 in the sera of cats for correlation analyses.

    Journal: Vaccines

    Article Title: Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

    doi: 10.3390/vaccines8040684

    Figure Lengend Snippet: Antibody responses in dogs and cats. ( A ) Serum antibody responses of dogs to S, S1, RBD, S2, N, E, and NS3 viral proteins of SARS-CoV-2, and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by IgG ELISA. The OD values were normalized by subtracting the OD values generated by sera from SPF dogs. ( B ) In canine serum samples, IgG levels against CCoV/HCoV and those against S, S1, RBD, S2, N, E, and NS3 of SARS-CoV-2, as detected by ELISA, were used in correlation analyses. ( C ) Serum IgG antibody responses of cats to SARS-CoV-2 S, S1, RBD, S2, N, E, and NS3, and and inactivated viruses, CCoV 1-71, HCoV NL63, and H1N1 influenza virus were measured by ELISA. The crude OD values generated by ELISA were normalized by subtracting the OD values generated by sera from SPF cats. ( D ) ELISA was also used to determine IgG antibody levels against N of SARS-CoV-2, CCoV 1-71, and HCoV NL63 in the sera of cats for correlation analyses.

    Article Snippet: Protein Microarray Assays Antigens printed onto microarrays were glycoproteins or nucleoproteins of HCoVs, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, respiratory syncytial viruses (RSVs), metapneumoviruses (MPVs), parainfluenza viruses (PIVs), adenoviruses (AdVs), and influenza viruses.

    Techniques: Enzyme-linked Immunosorbent Assay, Generated

    Preexisting and cross-reactive antibodies to human respiratory viruses in sera of patients with COVID-19. ( A ) Respiratory virus-specific antibody profiling. Heatmap indicating IgG antibody responses in 46 sera samples (37 COVID-19 samples and 9 control samples) measured by protein microarray. The microarray slides were probed with human sera and labeled with secondary antibodies to human IgG conjugated to a quantum dot fluorophore. The slides were imaged using the GenePix 4000B Microarray Scanner to measure background-subtracted median spot fluorescence. Mean fluorescence intensity (MFI) of the 4 replicates for each antigen was used for analysis. Each column represents a paired sample, and each row, a respiratory virus protein. Box graph indicates the sum of antibody responses of all sera samples for each respiratory virus protein. ( B ) Correlation between protein microarray and ELISA-based readouts. Each dot indicates the antibody response of serum from a single patient with COVID-19 against S, S1, RBD, S2, and N of SARS-CoV-2, as measured by fluorescence intensity (y-axis) and absorbance at 450 nm, as determined by ELISA (x-axis). ( C ) Relations of antibody response between specific antibody levels against SARS-CoV-2 and those against endemic HCoVs NL63, 229E, HKU1, and OC43 in patients with COVID-19.

    Journal: Vaccines

    Article Title: Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

    doi: 10.3390/vaccines8040684

    Figure Lengend Snippet: Preexisting and cross-reactive antibodies to human respiratory viruses in sera of patients with COVID-19. ( A ) Respiratory virus-specific antibody profiling. Heatmap indicating IgG antibody responses in 46 sera samples (37 COVID-19 samples and 9 control samples) measured by protein microarray. The microarray slides were probed with human sera and labeled with secondary antibodies to human IgG conjugated to a quantum dot fluorophore. The slides were imaged using the GenePix 4000B Microarray Scanner to measure background-subtracted median spot fluorescence. Mean fluorescence intensity (MFI) of the 4 replicates for each antigen was used for analysis. Each column represents a paired sample, and each row, a respiratory virus protein. Box graph indicates the sum of antibody responses of all sera samples for each respiratory virus protein. ( B ) Correlation between protein microarray and ELISA-based readouts. Each dot indicates the antibody response of serum from a single patient with COVID-19 against S, S1, RBD, S2, and N of SARS-CoV-2, as measured by fluorescence intensity (y-axis) and absorbance at 450 nm, as determined by ELISA (x-axis). ( C ) Relations of antibody response between specific antibody levels against SARS-CoV-2 and those against endemic HCoVs NL63, 229E, HKU1, and OC43 in patients with COVID-19.

    Article Snippet: Protein Microarray Assays Antigens printed onto microarrays were glycoproteins or nucleoproteins of HCoVs, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2, respiratory syncytial viruses (RSVs), metapneumoviruses (MPVs), parainfluenza viruses (PIVs), adenoviruses (AdVs), and influenza viruses.

    Techniques: Microarray, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay