sars cov sars cov 2 nucleocapsid antibody rabbit mab  (Sino Biological)


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    Name:
    SARS CoV SARS CoV 2 Nucleocapsid Antibody Rabbit MAb
    Description:
    This product is a recombinant monoclonal antibody expressed from HEK293 cells
    Catalog Number:
    40143-r001
    Product Aliases:
    Anti-coronavirus NP Antibody, Anti-coronavirus Nucleocapsid Antibody, Anti-coronavirus Nucleoprotein Antibody, Anti-cov np Antibody, Anti-ncov NP Antibody, Anti-novel coronavirus NP Antibody, Anti-novel coronavirus Nucleocapsid Antibody, Anti-novel coronavirus Nucleoprotein Antibody, Anti-NP Antibody, Anti-Nucleocapsid Antibody, Anti-Nucleoprotein Antibody
    Price:
    None
    Applications:
    WB,ELISA
    Host:
    Rabbit
    Immunogen:
    Recombinant SARS-CoV Nucleoprotein / NP Protein (Catalog#40143-V08B)
    Category:
    Primary Antibody
    Antibody Type:
    MAb
    Isotype:
    Rabbit IgG
    Reactivity:
    SARS
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    Structured Review

    Sino Biological sars cov sars cov 2 nucleocapsid antibody rabbit mab
    Binding of plant-produced monoclonal antibodies (mAbs) B38 and H4 to severe acute respiratory syndrome coronavirus 2 <t>(SARS-CoV-2)</t> receptor binding domain (RBD) protein. The ability of the plant-produced mAbs to recognize RBD protein of SARS-CoV-2 was assessed by ELISA. The plant-produced mAbs B38 and H4, standard human immunoglobulin G (IgG)1, and plant-produced anti- PD1 antibody (as negative control) ( Rattanapisit et al., 2019b ) were incubated on plates coated with commercial SARS-CoV-2 RBD. The bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-human kappa antibody. The data are the mean values of technical triplicates per concentration.
    This product is a recombinant monoclonal antibody expressed from HEK293 cells
    https://www.bioz.com/result/sars cov sars cov 2 nucleocapsid antibody rabbit mab/product/Sino Biological
    Average 94 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    sars cov sars cov 2 nucleocapsid antibody rabbit mab - by Bioz Stars, 2021-02
    94/100 stars

    Images

    1) Product Images from "Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro"

    Article Title: Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.589995

    Binding of plant-produced monoclonal antibodies (mAbs) B38 and H4 to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) protein. The ability of the plant-produced mAbs to recognize RBD protein of SARS-CoV-2 was assessed by ELISA. The plant-produced mAbs B38 and H4, standard human immunoglobulin G (IgG)1, and plant-produced anti- PD1 antibody (as negative control) ( Rattanapisit et al., 2019b ) were incubated on plates coated with commercial SARS-CoV-2 RBD. The bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-human kappa antibody. The data are the mean values of technical triplicates per concentration.
    Figure Legend Snippet: Binding of plant-produced monoclonal antibodies (mAbs) B38 and H4 to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) protein. The ability of the plant-produced mAbs to recognize RBD protein of SARS-CoV-2 was assessed by ELISA. The plant-produced mAbs B38 and H4, standard human immunoglobulin G (IgG)1, and plant-produced anti- PD1 antibody (as negative control) ( Rattanapisit et al., 2019b ) were incubated on plates coated with commercial SARS-CoV-2 RBD. The bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-human kappa antibody. The data are the mean values of technical triplicates per concentration.

    Techniques Used: Binding Assay, Produced, Enzyme-linked Immunosorbent Assay, Negative Control, Incubation, Concentration Assay

    2) 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

    3) Product Images from "Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes"

    Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

    Journal: bioRxiv

    doi: 10.1101/2021.01.17.427024

    SARS-CoV-2 Infects Astrocytes in Cortical Organoids A) Viral infection of cortical organoids. Human stem cells from several lines were aggregated and differentiated toward cortical identity in suspension. After 5, 10, 16 or 22 weeks of differentiation, organoids were exposed to SARS-CoV-2 for 2 hours, media was replaced and then collected 72 hours later. B) After 5, 10, or 16 weeks organoids only indicated rare infection (white arrowheads). At five and 10 weeks, SARS-CoV-2 N+ cells did not co-express SOX2, NEUN or GFAP indicating infected cells are not cortical progenitors, neurons or astrocytes and may instead be an off-target population. However, after 16 weeks, in one stem cell line a few GFAP+ astrocytes were infected. C) Although rare cells are infected at neurogenic stages, as indicated by coronavirus N antibody presence, there was no observed viral replication with dsRNA at these timepoints. D) At late gliogenic stages, by week 22 infection was readily observed in GFAP astrocytes but not NEUN+ neurons. E) 94% of infected cells stained positive for markers of astrocytes GFAP or AQP4, while only 4% are NEUN+ neurons. White arrowheads indicate SARS-CoV-2+ dsRNA+ GFAP+ AQP4+ astrocytes (GFAP+AQP4+ n=169 cells, NEUN n=143 cells).
    Figure Legend Snippet: SARS-CoV-2 Infects Astrocytes in Cortical Organoids A) Viral infection of cortical organoids. Human stem cells from several lines were aggregated and differentiated toward cortical identity in suspension. After 5, 10, 16 or 22 weeks of differentiation, organoids were exposed to SARS-CoV-2 for 2 hours, media was replaced and then collected 72 hours later. B) After 5, 10, or 16 weeks organoids only indicated rare infection (white arrowheads). At five and 10 weeks, SARS-CoV-2 N+ cells did not co-express SOX2, NEUN or GFAP indicating infected cells are not cortical progenitors, neurons or astrocytes and may instead be an off-target population. However, after 16 weeks, in one stem cell line a few GFAP+ astrocytes were infected. C) Although rare cells are infected at neurogenic stages, as indicated by coronavirus N antibody presence, there was no observed viral replication with dsRNA at these timepoints. D) At late gliogenic stages, by week 22 infection was readily observed in GFAP astrocytes but not NEUN+ neurons. E) 94% of infected cells stained positive for markers of astrocytes GFAP or AQP4, while only 4% are NEUN+ neurons. White arrowheads indicate SARS-CoV-2+ dsRNA+ GFAP+ AQP4+ astrocytes (GFAP+AQP4+ n=169 cells, NEUN n=143 cells).

    Techniques Used: Infection, Staining

    Coronavirus Protease Expression in Developing Human Cortex A) Single-cell RNA sequencing data from the developing cortex demonstrates minimal expression of canonical SARS-CoV-2 proteases TMPRSS2 and TMPRSS4 in cortical cell types. B) Alternative coronavirus proteases, TMPRSS5, FURIN and CTSB, are differentially expressed in a variety of cell types in the developing human cortex.
    Figure Legend Snippet: Coronavirus Protease Expression in Developing Human Cortex A) Single-cell RNA sequencing data from the developing cortex demonstrates minimal expression of canonical SARS-CoV-2 proteases TMPRSS2 and TMPRSS4 in cortical cell types. B) Alternative coronavirus proteases, TMPRSS5, FURIN and CTSB, are differentially expressed in a variety of cell types in the developing human cortex.

    Techniques Used: Expressing, RNA Sequencing Assay

    SARS-CoV-2 modestly infects other neural cell types A) Mature and precursor astroglial cells indicate high infection where 74% express PAX6, 56% express HOPX and 44% express S100B (PAX6 n=252 cells, HOPX n=405 cells, S100B n=343 across two biological samples and four technical replicates). B) Oligodendrocyte Precursor Cells (OPC) and Microglia have little expression compared to astrocytes (OLIG2 n=487 cells, IBA1 n=350 cells). C) Neurogenic intermediate progenitor cells (IPCs) also demonstrates minimal infection of excitatory neuron lineage (EOMES n=128 cells).
    Figure Legend Snippet: SARS-CoV-2 modestly infects other neural cell types A) Mature and precursor astroglial cells indicate high infection where 74% express PAX6, 56% express HOPX and 44% express S100B (PAX6 n=252 cells, HOPX n=405 cells, S100B n=343 across two biological samples and four technical replicates). B) Oligodendrocyte Precursor Cells (OPC) and Microglia have little expression compared to astrocytes (OLIG2 n=487 cells, IBA1 n=350 cells). C) Neurogenic intermediate progenitor cells (IPCs) also demonstrates minimal infection of excitatory neuron lineage (EOMES n=128 cells).

    Techniques Used: Infection, Expressing

    SARS-CoV-2 Infection Increases Cell Stress and Reactivity in Cortical Astrocytes A) After SAR-CoV-2 infection, there is no immediate increase in cell death in infected cells, as indicated by Cleaved Caspase 3 staining. B) Infected cells have an increase in cell stress, as indicated by the ER stress marker, ARCN1. C) Approximately one-third of infected astrocytes in primary cortical tissue express the reactive marker SYNM and three-quarters have the receptor for growth factor signaling, EGFR (SYNM n=62 cells, EGFR n=142 cells). D) The same proportion of infected organoid cells express SYNM and about one-half express EGFR (SYNM n=172 cells, EGFR n=143 cells).
    Figure Legend Snippet: SARS-CoV-2 Infection Increases Cell Stress and Reactivity in Cortical Astrocytes A) After SAR-CoV-2 infection, there is no immediate increase in cell death in infected cells, as indicated by Cleaved Caspase 3 staining. B) Infected cells have an increase in cell stress, as indicated by the ER stress marker, ARCN1. C) Approximately one-third of infected astrocytes in primary cortical tissue express the reactive marker SYNM and three-quarters have the receptor for growth factor signaling, EGFR (SYNM n=62 cells, EGFR n=142 cells). D) The same proportion of infected organoid cells express SYNM and about one-half express EGFR (SYNM n=172 cells, EGFR n=143 cells).

    Techniques Used: Infection, Staining, Marker

    Coronavirus Receptors, DPP4 and CD147, but not ACE2 are Expressed in Developing Human Cortex A) ACE2 expressing cells are readily infected by SARS-CoV-2, where they have abundant ACE2 and dsRNA presence. Primary cortical tissue and cortical organoids do not have observable ACE2 protein in cortical tissue or infected cells. NRP1 is present in cortical neurons, but not in infected cells. B) Infected cortical astrocytes in the SVZ of primary cortex abundantly express coronavirus receptors DPP4 and CD147, where 100% of cells assayed have DPP4 and 73% have CD147. In cortical organoids, 60% are DPP4+ and 28% are CD147+ (Primary: DPP4 n=61cells, CD147 n=83 cells; Organoid: DPP4 n=296 cells, CD147 n=239 cells) C) Inhibition of DPP4 by Vildagliptin results in a 30% decrease in the number of SARS-CoV-2 N+ cells and a 70% reduction in dsRNA+ cells. SARS-CoV-2 N+ dsRNA+ cells are indicated by white arrowheads (SARS-CoV-2 MOI 0.5 n=1273 cells, MOI 0.5+Vildagliptin n=879 cells, dsRNA MOI 0.5 n=571 cells, MOI 0.5+Vildagliptin n=227 cells across two biological samples and three technical replicates). D) ARCN1 is broadly expressed in SARS-CoV-2 infected samples, which is reduced by 70% after DPP4 inhibition by Vildagliptin. ARCN1+ dsRNA+ cells indicated by white arrowheads (MOI 0.5 n=1224 cells, MOI 0.5+ Vildagliptin n=331 cells across two biological samples and three technical replicates).
    Figure Legend Snippet: Coronavirus Receptors, DPP4 and CD147, but not ACE2 are Expressed in Developing Human Cortex A) ACE2 expressing cells are readily infected by SARS-CoV-2, where they have abundant ACE2 and dsRNA presence. Primary cortical tissue and cortical organoids do not have observable ACE2 protein in cortical tissue or infected cells. NRP1 is present in cortical neurons, but not in infected cells. B) Infected cortical astrocytes in the SVZ of primary cortex abundantly express coronavirus receptors DPP4 and CD147, where 100% of cells assayed have DPP4 and 73% have CD147. In cortical organoids, 60% are DPP4+ and 28% are CD147+ (Primary: DPP4 n=61cells, CD147 n=83 cells; Organoid: DPP4 n=296 cells, CD147 n=239 cells) C) Inhibition of DPP4 by Vildagliptin results in a 30% decrease in the number of SARS-CoV-2 N+ cells and a 70% reduction in dsRNA+ cells. SARS-CoV-2 N+ dsRNA+ cells are indicated by white arrowheads (SARS-CoV-2 MOI 0.5 n=1273 cells, MOI 0.5+Vildagliptin n=879 cells, dsRNA MOI 0.5 n=571 cells, MOI 0.5+Vildagliptin n=227 cells across two biological samples and three technical replicates). D) ARCN1 is broadly expressed in SARS-CoV-2 infected samples, which is reduced by 70% after DPP4 inhibition by Vildagliptin. ARCN1+ dsRNA+ cells indicated by white arrowheads (MOI 0.5 n=1224 cells, MOI 0.5+ Vildagliptin n=331 cells across two biological samples and three technical replicates).

    Techniques Used: Expressing, Infection, Inhibition

    SARS-CoV-2 Infects Astrocytes in Developing Human Cortex A) Experimental paradigm for viral infection of human cortical tissue. Primary cortical tissue was acutely sectioned and cultured at the air-liquid interface. The next day tissue was infected with SARS-CoV-2 at MOI 0.5 and cultured for 72 hours before being collected and processed. B) SARS-CoV-2 infects GFAP+AQP4+ astrocyte cells in the developing human cortex, which are predominantly located in the subventricular zone (SVZ), where > 81% of cells assayed expressed markers of astrocytes. 100% of infected cells expressed both SARS-CoV-2+ nucleocapsid (N) antibody and double-stranded (ds)RNA antibody. White arrowheads indicate dsRNA+GFAP+ infected astrocytes (dsRNA+SARS-CoV-2 N+ n=31 cells, GFAP+AQP4+ n=74 cells across two biological samples and four technical replicates). C) Few other neural types were infected, as indicated by co-labeling of SARS-CoV-2 N or dsRNA, where
    Figure Legend Snippet: SARS-CoV-2 Infects Astrocytes in Developing Human Cortex A) Experimental paradigm for viral infection of human cortical tissue. Primary cortical tissue was acutely sectioned and cultured at the air-liquid interface. The next day tissue was infected with SARS-CoV-2 at MOI 0.5 and cultured for 72 hours before being collected and processed. B) SARS-CoV-2 infects GFAP+AQP4+ astrocyte cells in the developing human cortex, which are predominantly located in the subventricular zone (SVZ), where > 81% of cells assayed expressed markers of astrocytes. 100% of infected cells expressed both SARS-CoV-2+ nucleocapsid (N) antibody and double-stranded (ds)RNA antibody. White arrowheads indicate dsRNA+GFAP+ infected astrocytes (dsRNA+SARS-CoV-2 N+ n=31 cells, GFAP+AQP4+ n=74 cells across two biological samples and four technical replicates). C) Few other neural types were infected, as indicated by co-labeling of SARS-CoV-2 N or dsRNA, where

    Techniques Used: Infection, Cell Culture, Labeling

    4) Product Images from "Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium"

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    Journal: bioRxiv

    doi: 10.1101/2020.08.27.271130

    A diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. ( A ) HAE-ALI model: Epithelial cells are taken from bronchia of the lung of healthy donors and plated onto Transwell ® inserts at an air-liquid interface (ALI) for four weeks. Four major types of the epithelial cells in the well differentiated polarized HAE cultures: basal, ciliated, goblet, and club cells are diagrammed in the Transwell ® insert, and their expression makers are indicated. ( B ) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CYKT5. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue). ( C ) Model of airway cell regeneration model of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, in which it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.
    Figure Legend Snippet: A diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. ( A ) HAE-ALI model: Epithelial cells are taken from bronchia of the lung of healthy donors and plated onto Transwell ® inserts at an air-liquid interface (ALI) for four weeks. Four major types of the epithelial cells in the well differentiated polarized HAE cultures: basal, ciliated, goblet, and club cells are diagrammed in the Transwell ® insert, and their expression makers are indicated. ( B ) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CYKT5. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue). ( C ) Model of airway cell regeneration model of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, in which it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.

    Techniques Used: Infection, Expressing, Staining

    Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE-ALI over a course of 21 days. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi). Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE-ALI over a course of 21 days. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi). Nuclei were stained with DAPI (blue).

    Techniques Used: Immunofluorescence, Infection, Staining

    SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV ( A ), and anti-MUC5AC ( B ), anti-cytokeratin 5 ( C ), and anti-SCGB1A1 ( D ), respectively. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV ( A ), and anti-MUC5AC ( B ), anti-cytokeratin 5 ( C ), and anti-SCGB1A1 ( D ), respectively. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue).

    Techniques Used: Infection, Staining

    Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).

    Techniques Used: Immunofluorescence, Infection, Staining

    Three-dimensional (z-stack) imaging of SARS-CoV-2 infected primary bronchial HAE-ALI. Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or with anti-NP and anti-β-tubulin IV antibodies (B), or co-stained anti-NP and anti-ZO-1 antibodies ( B ). A set of confocal images were taken at a magnification of x 40 from the stained pierce of epithelium at a distance of the Z value (μm), shown in each image, from the objective (z-axis) and reconstituted as a 3-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Three-dimensional (z-stack) imaging of SARS-CoV-2 infected primary bronchial HAE-ALI. Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or with anti-NP and anti-β-tubulin IV antibodies (B), or co-stained anti-NP and anti-ZO-1 antibodies ( B ). A set of confocal images were taken at a magnification of x 40 from the stained pierce of epithelium at a distance of the Z value (μm), shown in each image, from the objective (z-axis) and reconstituted as a 3-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).

    Techniques Used: Imaging, Infection, Staining, Fluorescence

    Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection). (A C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A), 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days post-infection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque forming units (pfu) were plotted to the DPI. Value represents the mean +/− standard deviations. ( B D ) Transepithelial electrical resistance measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set as 1.0. Values represent the mean of relative TEER +/− standard deviations. **** P
    Figure Legend Snippet: Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection). (A C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A), 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days post-infection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque forming units (pfu) were plotted to the DPI. Value represents the mean +/− standard deviations. ( B D ) Transepithelial electrical resistance measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set as 1.0. Values represent the mean of relative TEER +/− standard deviations. **** P

    Techniques Used: Infection, Incubation

    SARS-CoV-2 infection of primary human bronchial airway epithelium (HAE) is persistent. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque forming units (pfu) were determined (y-axis) and plotted to the dpi. Value represents the mean +/− standard deviations.
    Figure Legend Snippet: SARS-CoV-2 infection of primary human bronchial airway epithelium (HAE) is persistent. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque forming units (pfu) were determined (y-axis) and plotted to the dpi. Value represents the mean +/− standard deviations.

    Techniques Used: Infection

    SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2 infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque forming units (pfu) were plotted to the dpi. Value represents the mean +/− standard deviations. (B) Transepithelial electrical resistance (TEER) measurement. The TEER of infected HAE-ALI B4-20 cultures were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set as 1.0. Values represent the mean of the relative TEER +/− standard deviations. n.s. indicates statistically no significance. (C D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( C ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( D ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue)
    Figure Legend Snippet: SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2 infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque forming units (pfu) were plotted to the dpi. Value represents the mean +/− standard deviations. (B) Transepithelial electrical resistance (TEER) measurement. The TEER of infected HAE-ALI B4-20 cultures were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set as 1.0. Values represent the mean of the relative TEER +/− standard deviations. n.s. indicates statistically no significance. (C D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( C ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( D ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue)

    Techniques Used: Infection, Immunofluorescence, Staining

    5) Product Images from "Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium"

    Article Title: Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium

    Journal: mBio

    doi: 10.1128/mBio.02852-20

    Three-dimensional (z-stack) imaging of SARS-CoV-2-infected primary bronchial HAE-ALI. (A and B) Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were costained with anti-NP and anti-ZO-1 antibodies (A) or with anti-NP and anti-β-tubulin IV antibodies (B) or costained anti-NP and anti-ZO-1 antibodies (B). A set of confocal images were taken at a magnification of ×40 from the stained piece of epithelium at a distance of the Z value (in micrometers), shown in each image, from the objective ( z axis) and reconstituted as a three-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Three-dimensional (z-stack) imaging of SARS-CoV-2-infected primary bronchial HAE-ALI. (A and B) Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were costained with anti-NP and anti-ZO-1 antibodies (A) or with anti-NP and anti-β-tubulin IV antibodies (B) or costained anti-NP and anti-ZO-1 antibodies (B). A set of confocal images were taken at a magnification of ×40 from the stained piece of epithelium at a distance of the Z value (in micrometers), shown in each image, from the objective ( z axis) and reconstituted as a three-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).

    Techniques Used: Imaging, Infection, Staining, Fluorescence

    Immunofluorescence analysis of SARS-CoV-2-infected primary bronchial HAE-ALI over a course of 21 days. (A and B) Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were costained with anti-NP and anti-ZO-1 antibodies (A) or costained with anti-NP and anti-β-tubulin IV antibodies (B). Confocal images were taken at a magnification of ×40 on the indicated days postinfection (dpi). Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Immunofluorescence analysis of SARS-CoV-2-infected primary bronchial HAE-ALI over a course of 21 days. (A and B) Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were costained with anti-NP and anti-ZO-1 antibodies (A) or costained with anti-NP and anti-β-tubulin IV antibodies (B). Confocal images were taken at a magnification of ×40 on the indicated days postinfection (dpi). Nuclei were stained with DAPI (blue).

    Techniques Used: Immunofluorescence, Infection, Staining

    SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI = 0.2) were dissociated from the Transwell insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV (A), and anti-MUC5AC (B), anti-cytokeratin 5 (C), and anti-SCGB1A1 (D), respectively. Confocal images were taken at a magnification of ×63. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI = 0.2) were dissociated from the Transwell insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV (A), and anti-MUC5AC (B), anti-cytokeratin 5 (C), and anti-SCGB1A1 (D), respectively. Confocal images were taken at a magnification of ×63. Nuclei were stained with DAPI (blue).

    Techniques Used: Infection, Staining

    Immunofluorescence analysis of SARS-CoV-2-infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 2 × 10 −5 PFU/cell. (A and B) At 30 dpi, both virus- and mock-infected HAE were costained with anti-NP and anti-ZO-1 antibodies (A) or costained with anti-NP and anti-β-tubulin IV antibodies (B). Confocal images were taken at a magnification of ×40. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: Immunofluorescence analysis of SARS-CoV-2-infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 2 × 10 −5 PFU/cell. (A and B) At 30 dpi, both virus- and mock-infected HAE were costained with anti-NP and anti-ZO-1 antibodies (A) or costained with anti-NP and anti-β-tubulin IV antibodies (B). Confocal images were taken at a magnification of ×40. Nuclei were stained with DAPI (blue).

    Techniques Used: Immunofluorescence, Infection, Staining

    Diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. (A) HAE-ALI model. Epithelial cells are taken from bronchia from the lungs of healthy donors and plated onto Transwell inserts at an air-liquid interface (ALI) for 4 weeks. Four major types of epithelial cells in the well-differentiated polarized HAE cultures, basal, ciliated, goblet, and club cells, are diagrammed in the Transwell insert, and their expression markers are indicated. (B) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI = 0.2) were dissociated from the Transwell insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CKRT5. Confocal images were taken at a magnification of ×63. Nuclei were stained with DAPI (blue). (C) Model of airway cell regeneration of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, where it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.
    Figure Legend Snippet: Diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. (A) HAE-ALI model. Epithelial cells are taken from bronchia from the lungs of healthy donors and plated onto Transwell inserts at an air-liquid interface (ALI) for 4 weeks. Four major types of epithelial cells in the well-differentiated polarized HAE cultures, basal, ciliated, goblet, and club cells, are diagrammed in the Transwell insert, and their expression markers are indicated. (B) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI = 0.2) were dissociated from the Transwell insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CKRT5. Confocal images were taken at a magnification of ×63. Nuclei were stained with DAPI (blue). (C) Model of airway cell regeneration of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, where it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.

    Techniques Used: Infection, Expressing, Staining

    SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2-infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque-forming units (PFU) were plotted to the dpi. Value represent means ± standard deviations. (B) TEER measurement. The TEER of infected HAE-ALI B4-20 cultures was measured using an epithelial volt-ohm meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set at 1.0. Values represent means of the relative TEER ± standard deviations. n.s., statistically not significant. (C and D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were costained with anti-NP and anti-ZO-1 antibodies (C) or costained with anti-NP and anti-β-tubulin IV antibodies (D). Confocal images were taken at a magnification of ×40. Nuclei were stained with DAPI (blue). Basol, basolateral.
    Figure Legend Snippet: SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2-infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque-forming units (PFU) were plotted to the dpi. Value represent means ± standard deviations. (B) TEER measurement. The TEER of infected HAE-ALI B4-20 cultures was measured using an epithelial volt-ohm meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set at 1.0. Values represent means of the relative TEER ± standard deviations. n.s., statistically not significant. (C and D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were costained with anti-NP and anti-ZO-1 antibodies (C) or costained with anti-NP and anti-β-tubulin IV antibodies (D). Confocal images were taken at a magnification of ×40. Nuclei were stained with DAPI (blue). Basol, basolateral.

    Techniques Used: Infection, Immunofluorescence, Staining

    SARS-CoV-2 replication in primary human bronchial airway epithelium (HAE) over a course of 21 days. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days postinfection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque-forming units (PFU) were determined ( y axis) and plotted to the day postinfection. Values represent means ± standard deviations (SD) (error bars).
    Figure Legend Snippet: SARS-CoV-2 replication in primary human bronchial airway epithelium (HAE) over a course of 21 days. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days postinfection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque-forming units (PFU) were determined ( y axis) and plotted to the day postinfection. Values represent means ± standard deviations (SD) (error bars).

    Techniques Used: Infection

    Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection [MOIs]). (A and C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A) and 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days postinfection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque-forming units (PFU) were plotted to the dpi. Values represent means ± standard deviations. (B and D) TEER measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture was measured using an epithelial volt-ohm meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set at 1.0. Values represent the means of relative TEER ± standard deviations. *** * , P
    Figure Legend Snippet: Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection [MOIs]). (A and C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A) and 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days postinfection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque-forming units (PFU) were plotted to the dpi. Values represent means ± standard deviations. (B and D) TEER measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture was measured using an epithelial volt-ohm meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set at 1.0. Values represent the means of relative TEER ± standard deviations. *** * , P

    Techniques Used: Infection, Incubation

    6) Product Images from "SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring"

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring

    Journal: Matter

    doi: 10.1016/j.matt.2020.09.027

    Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).

    Techniques Used: Construct

    Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).
    Figure Legend Snippet: Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Whisker Assay

    7) Product Images from "Interplay of Antibody and Cytokine Production Reveals CXCL13 as a Potential Novel Biomarker of Lethal SARS-CoV-2 Infection"

    Article Title: Interplay of Antibody and Cytokine Production Reveals CXCL13 as a Potential Novel Biomarker of Lethal SARS-CoV-2 Infection

    Journal: mSphere

    doi: 10.1128/mSphere.01324-20

    Anti-SARS-CoV-2 IgG response of SARS-CoV-2 inpatients. Antibody (IgG) levels of patient samples that tested PCR positive (red) or negative (clear) for SARS-CoV-2 to RBD (a), N (b), or S1 (c). Correlation of antibody levels to RBD versus N (d) or S1 (e). Correlation of antibody levels to N versus S1 (f). Antibody levels of anti-RBD (g), anti-N (h), or anti-S1 (i) produced by SARS-CoV-2-positive patients versus days after SARS-CoV-2 disease onset. n = 491 patient samples.
    Figure Legend Snippet: Anti-SARS-CoV-2 IgG response of SARS-CoV-2 inpatients. Antibody (IgG) levels of patient samples that tested PCR positive (red) or negative (clear) for SARS-CoV-2 to RBD (a), N (b), or S1 (c). Correlation of antibody levels to RBD versus N (d) or S1 (e). Correlation of antibody levels to N versus S1 (f). Antibody levels of anti-RBD (g), anti-N (h), or anti-S1 (i) produced by SARS-CoV-2-positive patients versus days after SARS-CoV-2 disease onset. n = 491 patient samples.

    Techniques Used: Polymerase Chain Reaction, Produced

    CXCL13 as a novel biomarker for lethal SARS-CoV-2 infection. CXCL13 peak (a) or average (b) concentration was measured in SARS-CoV-2-positive and -negative patients. CXCL13 production by SARS-CoV-2 production is compared to anti-RBD (c) or anti-N (d) IgG quantity over the course of patient disease. Examples of a surviving patient producing low CXCL13 and low anti-RBD IgG response (e) or deceased patient producing high CXCL13 and high anti-RBD IgG response (f). Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. *, P
    Figure Legend Snippet: CXCL13 as a novel biomarker for lethal SARS-CoV-2 infection. CXCL13 peak (a) or average (b) concentration was measured in SARS-CoV-2-positive and -negative patients. CXCL13 production by SARS-CoV-2 production is compared to anti-RBD (c) or anti-N (d) IgG quantity over the course of patient disease. Examples of a surviving patient producing low CXCL13 and low anti-RBD IgG response (e) or deceased patient producing high CXCL13 and high anti-RBD IgG response (f). Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. *, P

    Techniques Used: Biomarker Assay, Infection, Concentration Assay

    8) Product Images from "Neuropathology of patients with COVID-19 in Germany: a post-mortem case series"

    Article Title: Neuropathology of patients with COVID-19 in Germany: a post-mortem case series

    Journal: The Lancet. Neurology

    doi: 10.1016/S1474-4422(20)30308-2

    Neuropathological findings and SARS-CoV-2 viral loads in studied patients (n=43) Cases are arranged from left to right on the basis of the presence and quantity of SARS-CoV-2 in the brain. F=female. FFPE=formalin-fixed paraffin-embedded. HPF=high-power field. IHC=immunohistochemistry. M=male. P=parenchymal. PV=perivascular. qPCR=quantitative PCR. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. *Values shown for positive cases represent number of copies of SARS-CoV-2 RNA (× 10 3 /mL); detection was done in the frontal lobe in cryopreserved specimens and in the upper medulla oblongata in FFPE specimens.
    Figure Legend Snippet: Neuropathological findings and SARS-CoV-2 viral loads in studied patients (n=43) Cases are arranged from left to right on the basis of the presence and quantity of SARS-CoV-2 in the brain. F=female. FFPE=formalin-fixed paraffin-embedded. HPF=high-power field. IHC=immunohistochemistry. M=male. P=parenchymal. PV=perivascular. qPCR=quantitative PCR. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. *Values shown for positive cases represent number of copies of SARS-CoV-2 RNA (× 10 3 /mL); detection was done in the frontal lobe in cryopreserved specimens and in the upper medulla oblongata in FFPE specimens.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Real-time Polymerase Chain Reaction

    Distribution of SARS-CoV-2 within the CNS Representative images of viral protein-positive cells (green arrows) in the medulla oblongata detected by anti-nucleocapsid protein antibody (A) or anti-spike protein antibody (B). (C) SARS-CoV-2 nucleoprotein (brown staining ) could also be detected in subsets of cranial nerves originating from the lower brainstem. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Distribution of SARS-CoV-2 within the CNS Representative images of viral protein-positive cells (green arrows) in the medulla oblongata detected by anti-nucleocapsid protein antibody (A) or anti-spike protein antibody (B). (C) SARS-CoV-2 nucleoprotein (brown staining ) could also be detected in subsets of cranial nerves originating from the lower brainstem. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.

    Techniques Used: Staining

    9) Product Images from "SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring"

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring

    Journal: Matter

    doi: 10.1016/j.matt.2020.09.027

    Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).

    Techniques Used: Construct

    Investigation of the Selectivity and Multiplexed Performance of the Wireless SARS-CoV-2 RapidPlex Platform (A) Selective response of NP, S1-IgG and S1-IgM isotypes, and CRP sensors against different non-target circulating analytes. Interferential molecules were tested at 500 pg mL −1 (with an exception of 50 ng mL −1 for CRP), 250 ng mL −1 , and 50 ng mL −1 for NP, S1-IgG and S1-IgM, and CRP assays, respectively. Data are presented as mean ± SD (n = 3). (B) Validation of sample concentrations measured using the designed electrochemical sensor against sample concentrations measured using ELISA. (C) Block diagram of the SARS-CoV-2 RapidPlex platform. UART, universal asynchronous receiver/transmitter; MCU, microcontroller unit; DAC, digital-to-analog converter; ADC, analog-to-digital converter. (D) Schematic illustration of the graphene sensor array layout. (E) Experimental readings obtained with the functionalized SARS-CoV-2 RapidPlex platform after incubation of the four WEs with PBS (pH 7.4) supplemented with 1.0% BSA containing 1.0 ng mL −1 NP (I), 250 ng mL −1 S1-IgG (II), 250 ng mL −1 S1-IgM (III), and 50 ng mL −1 CRP (IV).
    Figure Legend Snippet: Investigation of the Selectivity and Multiplexed Performance of the Wireless SARS-CoV-2 RapidPlex Platform (A) Selective response of NP, S1-IgG and S1-IgM isotypes, and CRP sensors against different non-target circulating analytes. Interferential molecules were tested at 500 pg mL −1 (with an exception of 50 ng mL −1 for CRP), 250 ng mL −1 , and 50 ng mL −1 for NP, S1-IgG and S1-IgM, and CRP assays, respectively. Data are presented as mean ± SD (n = 3). (B) Validation of sample concentrations measured using the designed electrochemical sensor against sample concentrations measured using ELISA. (C) Block diagram of the SARS-CoV-2 RapidPlex platform. UART, universal asynchronous receiver/transmitter; MCU, microcontroller unit; DAC, digital-to-analog converter; ADC, analog-to-digital converter. (D) Schematic illustration of the graphene sensor array layout. (E) Experimental readings obtained with the functionalized SARS-CoV-2 RapidPlex platform after incubation of the four WEs with PBS (pH 7.4) supplemented with 1.0% BSA containing 1.0 ng mL −1 NP (I), 250 ng mL −1 S1-IgG (II), 250 ng mL −1 S1-IgM (III), and 50 ng mL −1 CRP (IV).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation

    Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).
    Figure Legend Snippet: Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Whisker Assay

    A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS-CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.
    Figure Legend Snippet: A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS-CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.

    Techniques Used: Multiplex Assay, Biomarker Assay

    Characterization of Electrochemical Graphene Biosensors Comprising the SARS-CoV-2 RapidPlex Platform (A) Scheme detailing the methodology developed for the covalent attachment of the corresponding bioreceptor for the specific capture of the target analytes SARS-CoV-2 NP and CRP (left), and IgG and IgM isotypes against SARS-CoV-2 S1 protein (right). PBA, 1-pyrenebutyric acid; BSA, bovine serum albumin; CAb, capture antibody; PI, polyimide. (B and C) Differential pulse voltammetry (B) and Nyquist plots (C) of a graphene electrode in 0.01 M PBS (pH 7.4) containing 2.0 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1) after each modification step (S1-IgG assay as representative example): bare graphene (Bare), functionalization with PBA (PBA), immobilization of SARS-CoV-2 S1 protein (Protein), blocking with BSA (BSA), recognition of specific S1-IgG (Target), and incubation with enzyme-tagged anti-human IgG antibody (DAb). (D) Comparison of amperometric responses and overlaid signal-to-blank (S/B) ratio (black lines) for SARS-CoV-2-specific IgG and CRP detection using PBA and 1H-pyrrole-1-propionic acid (PPA) as linkers for the attachment of the corresponding capture bioreceptors. Data are presented as mean ± SD (n = 3). (E) Amperometric responses and overlaid S/B ratio (black lines) observed for 0.0 and 500 pg mL −1 NP, 0.0 and 250 ng mL −1 SARS-CoV-2-specific IgG and IgM, and 0.0 and 50 ng mL −1 CRP, with 1-, 5-, and 10-min incubation. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Characterization of Electrochemical Graphene Biosensors Comprising the SARS-CoV-2 RapidPlex Platform (A) Scheme detailing the methodology developed for the covalent attachment of the corresponding bioreceptor for the specific capture of the target analytes SARS-CoV-2 NP and CRP (left), and IgG and IgM isotypes against SARS-CoV-2 S1 protein (right). PBA, 1-pyrenebutyric acid; BSA, bovine serum albumin; CAb, capture antibody; PI, polyimide. (B and C) Differential pulse voltammetry (B) and Nyquist plots (C) of a graphene electrode in 0.01 M PBS (pH 7.4) containing 2.0 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1) after each modification step (S1-IgG assay as representative example): bare graphene (Bare), functionalization with PBA (PBA), immobilization of SARS-CoV-2 S1 protein (Protein), blocking with BSA (BSA), recognition of specific S1-IgG (Target), and incubation with enzyme-tagged anti-human IgG antibody (DAb). (D) Comparison of amperometric responses and overlaid signal-to-blank (S/B) ratio (black lines) for SARS-CoV-2-specific IgG and CRP detection using PBA and 1H-pyrrole-1-propionic acid (PPA) as linkers for the attachment of the corresponding capture bioreceptors. Data are presented as mean ± SD (n = 3). (E) Amperometric responses and overlaid S/B ratio (black lines) observed for 0.0 and 500 pg mL −1 NP, 0.0 and 250 ng mL −1 SARS-CoV-2-specific IgG and IgM, and 0.0 and 50 ng mL −1 CRP, with 1-, 5-, and 10-min incubation. Data are presented as mean ± SD (n = 3).

    Techniques Used: Modification, Blocking Assay, Incubation

    10) Product Images from "Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues"

    Article Title: Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues

    Journal: Applied Immunohistochemistry & Molecular Morphology

    doi: 10.1097/PAI.0000000000000878

    H E stain (A), in situ hybridization (B) and immunohistochemical staining (C–H) of SARS-CoV2 positive lung (A–E) and SARS-CoV2 negative tissues (F–H); ISH probe to S1 spike protein (B) demonstrating SARS-CoV2 viral RNA in lung tissue(B); SARS-CoV2-positive lung tissues negative for mAb FIPV3-20 (C) and mAb 007 (D); mAb 019, intense immunolabelling of hyaline membranes in lung (E), extensive nonspecific immunolabelling of skin (F), colon (G), and spleen (H).
    Figure Legend Snippet: H E stain (A), in situ hybridization (B) and immunohistochemical staining (C–H) of SARS-CoV2 positive lung (A–E) and SARS-CoV2 negative tissues (F–H); ISH probe to S1 spike protein (B) demonstrating SARS-CoV2 viral RNA in lung tissue(B); SARS-CoV2-positive lung tissues negative for mAb FIPV3-20 (C) and mAb 007 (D); mAb 019, intense immunolabelling of hyaline membranes in lung (E), extensive nonspecific immunolabelling of skin (F), colon (G), and spleen (H).

    Techniques Used: Staining, In Situ Hybridization, Immunohistochemistry

    Pellets of HEK293 cells transfected with SARS-CoV2: S1 and S2 subunit of spike protein, nucleoprotein and untransfected cells. A–D, Hematoxylin eosin stain/H E. E–H, mAb FIPV3-20 to nucleoprotein, no immunolabeling of any pellet. I–L, mAb 019, intense immunostaining of all cell pellets. M–P, mAb 1A9, exclusive immunoreactivity of HEK293 cells transfected with spike protein S2 subunit. Q–T, mAb 001, homogeneous staining of HEK293 cells expressing nucleoprotein. U–X. In situ hybridization with probe to S1 subunit positive in corresponding HEK293 cells.
    Figure Legend Snippet: Pellets of HEK293 cells transfected with SARS-CoV2: S1 and S2 subunit of spike protein, nucleoprotein and untransfected cells. A–D, Hematoxylin eosin stain/H E. E–H, mAb FIPV3-20 to nucleoprotein, no immunolabeling of any pellet. I–L, mAb 019, intense immunostaining of all cell pellets. M–P, mAb 1A9, exclusive immunoreactivity of HEK293 cells transfected with spike protein S2 subunit. Q–T, mAb 001, homogeneous staining of HEK293 cells expressing nucleoprotein. U–X. In situ hybridization with probe to S1 subunit positive in corresponding HEK293 cells.

    Techniques Used: Transfection, Staining, Immunolabeling, Immunostaining, Expressing, In Situ Hybridization

    Analysis of autopsy tissue from patients who died from COVID-19: H E stain (A, D) immunoreactivity with mAb 1A9 to S2 subunit spike protein (B, E) and mAb 001 to nucleoprotein (C, F–I); COVID-19 pneumonia in acute phase diffuse alveolar damage with serial sections stained with H E (A), mAb 1A9 (B), and mAb 001 (C); extensive immunostaining of hyaline membranes and alveolar macrophages for both spike protein and nucleoprotein. mAb 001 staining in endothelia of focal septal vessels and pneumocytes (inset); mucus filled bronchus (D) H E stain with weakly positive S2 spike protein content (E) and strong signal for nucleoprotein (F); heart muscle with mAb 001 immunopositive endothelia; mAb 001/nucleoprotein-positive content in tubule of SARS-CoV2-positive autopsy kidney (H) and smear of nasal swab stained with mAb 001 positive for SARS-CoV2 (I).
    Figure Legend Snippet: Analysis of autopsy tissue from patients who died from COVID-19: H E stain (A, D) immunoreactivity with mAb 1A9 to S2 subunit spike protein (B, E) and mAb 001 to nucleoprotein (C, F–I); COVID-19 pneumonia in acute phase diffuse alveolar damage with serial sections stained with H E (A), mAb 1A9 (B), and mAb 001 (C); extensive immunostaining of hyaline membranes and alveolar macrophages for both spike protein and nucleoprotein. mAb 001 staining in endothelia of focal septal vessels and pneumocytes (inset); mucus filled bronchus (D) H E stain with weakly positive S2 spike protein content (E) and strong signal for nucleoprotein (F); heart muscle with mAb 001 immunopositive endothelia; mAb 001/nucleoprotein-positive content in tubule of SARS-CoV2-positive autopsy kidney (H) and smear of nasal swab stained with mAb 001 positive for SARS-CoV2 (I).

    Techniques Used: Staining, Immunostaining

    11) Product Images from "SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis"

    Article Title: SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis

    Journal: bioRxiv

    doi: 10.1101/2020.11.04.364315

    RNA sequencing identified robust viral transcription and activation of innate immune response in hPSC-derived cardiomyocytes and 2D tissues. a, MDS plot of RNA sequencing data obtained from mock and SARS-CoV-2 infected (MOI 0.1) hPSC-derived cardiomyocytes (CM), fibroblasts (Fb), macrophages (Mac), and two-dimensional tissues (CM+Fb+Mac) containing all 3 cellular components. Cells and tissues were harvested on day 3 post-inoculation. n=5 per experimental group. b, Heatmap of SARS-CoV-2 viral gene expression in each condition. Color scale denotes absolute expression as log2 of counts per million reads (CPM) (blue=0, red=15). c, Volcano plots showing differentially expressed genes between mock and SARS-CoV-2 infected conditions. Black: no significant change, Red: upregulated during infection (log2 fold change > 2, FDR p-value
    Figure Legend Snippet: RNA sequencing identified robust viral transcription and activation of innate immune response in hPSC-derived cardiomyocytes and 2D tissues. a, MDS plot of RNA sequencing data obtained from mock and SARS-CoV-2 infected (MOI 0.1) hPSC-derived cardiomyocytes (CM), fibroblasts (Fb), macrophages (Mac), and two-dimensional tissues (CM+Fb+Mac) containing all 3 cellular components. Cells and tissues were harvested on day 3 post-inoculation. n=5 per experimental group. b, Heatmap of SARS-CoV-2 viral gene expression in each condition. Color scale denotes absolute expression as log2 of counts per million reads (CPM) (blue=0, red=15). c, Volcano plots showing differentially expressed genes between mock and SARS-CoV-2 infected conditions. Black: no significant change, Red: upregulated during infection (log2 fold change > 2, FDR p-value

    Techniques Used: RNA Sequencing Assay, Activation Assay, Derivative Assay, Infection, Expressing

    Human engineered heart tissues (EHTs) recapitulate aspects of COVID-19 myocarditis. a, Representative hematoxylin and eosin (H E) stained histology images of three-dimensional EHTs consisting of hPSC-derived cardiomyocytes (CM), fibroblasts (Fb), and macrophages (Macs) 5 days following mock infection or inoculation with SARS-CoV-2 (MOI 0.1). Insets are high magnification images of the boxed areas. Representative images from 4 independent samples. b , Immunostaining of mock or SARS-CoV-2 infected three-dimensional EHTs for sarcomeric actin (cardiomyocytes, red), CD68 (macrophages, green), and nucleocapsid protein (white). EHTs were harvested 5 days after inoculation. Blue: DAPI. Images are representative of 4 independent experiments. Representative images from 4 independent samples. c, Quantitative RT-PCR of SARS-CoV-2 N gene expression in EHTs consisting of hPSC-derived cardiomyocytes (CM) and fibroblasts (Fb) or hPSC-derived cardiomyocytes, fibroblasts, and macrophages. EHTs were either mock infected or inoculated with SARS-CoV-2 (MOI 0.1) and harvested 5 days after inoculation. Each data point represents individual samples/experiments. Error bars denote standard error of the mean. Bar height represents sample mean. Dotted line: limit of detection. *p
    Figure Legend Snippet: Human engineered heart tissues (EHTs) recapitulate aspects of COVID-19 myocarditis. a, Representative hematoxylin and eosin (H E) stained histology images of three-dimensional EHTs consisting of hPSC-derived cardiomyocytes (CM), fibroblasts (Fb), and macrophages (Macs) 5 days following mock infection or inoculation with SARS-CoV-2 (MOI 0.1). Insets are high magnification images of the boxed areas. Representative images from 4 independent samples. b , Immunostaining of mock or SARS-CoV-2 infected three-dimensional EHTs for sarcomeric actin (cardiomyocytes, red), CD68 (macrophages, green), and nucleocapsid protein (white). EHTs were harvested 5 days after inoculation. Blue: DAPI. Images are representative of 4 independent experiments. Representative images from 4 independent samples. c, Quantitative RT-PCR of SARS-CoV-2 N gene expression in EHTs consisting of hPSC-derived cardiomyocytes (CM) and fibroblasts (Fb) or hPSC-derived cardiomyocytes, fibroblasts, and macrophages. EHTs were either mock infected or inoculated with SARS-CoV-2 (MOI 0.1) and harvested 5 days after inoculation. Each data point represents individual samples/experiments. Error bars denote standard error of the mean. Bar height represents sample mean. Dotted line: limit of detection. *p

    Techniques Used: Staining, Derivative Assay, Magnetic Cell Separation, Infection, Immunostaining, Quantitative RT-PCR, Expressing

    SARS-CoV-2 entry of hPSC-derived cardiomyocytes is mediated by ACE2 and endosomal cysteine proteases. a-b, hPSC-derived cardiomyocytes were infected with mock or inoculated with SARS-CoV-2-mNeonGreen (MOI 0.1). Cells were treated with either vehicle control, anti-human ACE2 neutralizing antibody (Anti-hACE2 mAb, left), or remdesivir (inhibitor of RNA-dependent RNA polymerase, right) at the indicated concentrations. Cells were analyzed by flow cytometry on day 3 post-inoculation for viral infection (NeonGreen, green circles) and viability (Zombie-Violet, black circles) ( a ). The presence of viral RNA in the tissue culture supernatant was also quantified by RT-PCR ( b ). Each data point corresponds to an individual sample/experiment, error bars denote standard error of the mean, *p
    Figure Legend Snippet: SARS-CoV-2 entry of hPSC-derived cardiomyocytes is mediated by ACE2 and endosomal cysteine proteases. a-b, hPSC-derived cardiomyocytes were infected with mock or inoculated with SARS-CoV-2-mNeonGreen (MOI 0.1). Cells were treated with either vehicle control, anti-human ACE2 neutralizing antibody (Anti-hACE2 mAb, left), or remdesivir (inhibitor of RNA-dependent RNA polymerase, right) at the indicated concentrations. Cells were analyzed by flow cytometry on day 3 post-inoculation for viral infection (NeonGreen, green circles) and viability (Zombie-Violet, black circles) ( a ). The presence of viral RNA in the tissue culture supernatant was also quantified by RT-PCR ( b ). Each data point corresponds to an individual sample/experiment, error bars denote standard error of the mean, *p

    Techniques Used: Derivative Assay, Infection, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

    Human autopsy and endomyocardial tissue from patients with suspected COVID-19 myocarditis show evidence of SARS-CoV-2 cardiomyocyte infection. a , Hematoxylin and eosin staining of cardiac autopsy (anterior left ventricular wall) and biopsy samples (right ventricular septum) from subjects without COVID-19 (control case) and patients with a clinical diagnosis of COVID-19 myocarditis (case 1-4). b, In situ hybridization of cardiac autopsy and biopsy tissue for SARS-CoV-2 spike and nucleocapsid RNA (red) showing evidence of viral infection. Hematoxylin: blue. Arrows denotes viral RNA staining in cells with cardiomyocyte morphology. c , Immunostaining of control and COVID-19 myocarditis cardiac autopsy tissue for SARS-CoV-2 nucleocapsid (white) and cardiac actin (red). DAPI: blue. Arrows denotes nucleocapsid staining in cardiomyocytes. d, Immunostaining of control and COVID-19 myocarditis cardiac autopsy and biopsy tissue for CD68 (green) and CCR2 (red). DAPI: blue. e, Immunostaining of control and COVID-19 myocarditis cardiac autopsy and biopsy tissue for CD3 (brown). Hematoxylin: blue.
    Figure Legend Snippet: Human autopsy and endomyocardial tissue from patients with suspected COVID-19 myocarditis show evidence of SARS-CoV-2 cardiomyocyte infection. a , Hematoxylin and eosin staining of cardiac autopsy (anterior left ventricular wall) and biopsy samples (right ventricular septum) from subjects without COVID-19 (control case) and patients with a clinical diagnosis of COVID-19 myocarditis (case 1-4). b, In situ hybridization of cardiac autopsy and biopsy tissue for SARS-CoV-2 spike and nucleocapsid RNA (red) showing evidence of viral infection. Hematoxylin: blue. Arrows denotes viral RNA staining in cells with cardiomyocyte morphology. c , Immunostaining of control and COVID-19 myocarditis cardiac autopsy tissue for SARS-CoV-2 nucleocapsid (white) and cardiac actin (red). DAPI: blue. Arrows denotes nucleocapsid staining in cardiomyocytes. d, Immunostaining of control and COVID-19 myocarditis cardiac autopsy and biopsy tissue for CD68 (green) and CCR2 (red). DAPI: blue. e, Immunostaining of control and COVID-19 myocarditis cardiac autopsy and biopsy tissue for CD3 (brown). Hematoxylin: blue.

    Techniques Used: Infection, Staining, In Situ Hybridization, Immunostaining

    SARS-CoV-2 infects cardiomyocytes. a , Focus forming assay demonstrating production of infectious virus from cultures containing hPSC-derived cardiomyocytes (CM), fibroblasts (Fb), and macrophages (Mac) inoculated with SARS-CoV-2 (MOI 0.1). Media only denotes wells that contain no cells. Assays were performed 3 days following inoculation. Dashed line shows the limit of assay detection. b , Quantitative RT-PCR showing viral N gene copies in cultures containing CM, Fb, and Macs inoculated with SARS-CoV-2 (MOI 0.1). RNA was collected 3 days after inoculation. Data points indicate individual samples (n=5, a-b). Bars denote the mean value and error bars indicate standard error of the mean. c , Focus forming assay measuring infectious SARS-CoV-2 (wild-type, black; mNeonGreen, green) in supernatant of hPSC-derived cardiomyocytes over time following inoculation (MOI 0.1). Dashed line shows the limit of detection. n=4 per experimental group. Error bars denote standard deviation. d , Two-dimensional cultures of hPSC-derived cardiomyocytes were inoculated with SARS-CoV-2 (MOI 0.1) and analyzed for viability (Zombie-Violet) and infection (NeonGreen reporter) as a function of time by flow cytometry. Right plot: viability of NeonGreen positive cells. n=4 per experimental group. Error bars denote standard deviation. e , Brightfield microscopy showing cytopathic effect (CPE) in hPSC-derived cardiomyocytes infected with SARS-CoV-2 (MOI 0.1). Representative images from 5 individual samples. f , Flow cytometry of two-dimensional tissues containing CM and Fb (left) or CM, Fb, and Mac (right) harvested on day 3 following either mock infection or inoculation with SARS-CoV-2 (MOI 0.1). Representative plot from 4 independent samples. Cardiomyocytes (CD90-CD14−) demonstrated prominent NeonGreen fluorescence (green overlay). NeonGreen signal was not detected in fibroblasts (CD90+CD14−) or macrophages (CD90-CD14+). g , Quantification of the percent NeonGreen positive cells from 2-dimensional tissues containing hPSC-CMs and fibroblasts or hPSC-CMs, fibroblasts, and macrophages. Data points indicate individual samples (n=4). * p
    Figure Legend Snippet: SARS-CoV-2 infects cardiomyocytes. a , Focus forming assay demonstrating production of infectious virus from cultures containing hPSC-derived cardiomyocytes (CM), fibroblasts (Fb), and macrophages (Mac) inoculated with SARS-CoV-2 (MOI 0.1). Media only denotes wells that contain no cells. Assays were performed 3 days following inoculation. Dashed line shows the limit of assay detection. b , Quantitative RT-PCR showing viral N gene copies in cultures containing CM, Fb, and Macs inoculated with SARS-CoV-2 (MOI 0.1). RNA was collected 3 days after inoculation. Data points indicate individual samples (n=5, a-b). Bars denote the mean value and error bars indicate standard error of the mean. c , Focus forming assay measuring infectious SARS-CoV-2 (wild-type, black; mNeonGreen, green) in supernatant of hPSC-derived cardiomyocytes over time following inoculation (MOI 0.1). Dashed line shows the limit of detection. n=4 per experimental group. Error bars denote standard deviation. d , Two-dimensional cultures of hPSC-derived cardiomyocytes were inoculated with SARS-CoV-2 (MOI 0.1) and analyzed for viability (Zombie-Violet) and infection (NeonGreen reporter) as a function of time by flow cytometry. Right plot: viability of NeonGreen positive cells. n=4 per experimental group. Error bars denote standard deviation. e , Brightfield microscopy showing cytopathic effect (CPE) in hPSC-derived cardiomyocytes infected with SARS-CoV-2 (MOI 0.1). Representative images from 5 individual samples. f , Flow cytometry of two-dimensional tissues containing CM and Fb (left) or CM, Fb, and Mac (right) harvested on day 3 following either mock infection or inoculation with SARS-CoV-2 (MOI 0.1). Representative plot from 4 independent samples. Cardiomyocytes (CD90-CD14−) demonstrated prominent NeonGreen fluorescence (green overlay). NeonGreen signal was not detected in fibroblasts (CD90+CD14−) or macrophages (CD90-CD14+). g , Quantification of the percent NeonGreen positive cells from 2-dimensional tissues containing hPSC-CMs and fibroblasts or hPSC-CMs, fibroblasts, and macrophages. Data points indicate individual samples (n=4). * p

    Techniques Used: Focus Forming Assay, Derivative Assay, Quantitative RT-PCR, Magnetic Cell Separation, Standard Deviation, Infection, Flow Cytometry, Microscopy, Fluorescence

    Mechanisms of reduced EHT contractility. a , Combined immunostaining for cardiomyocytes (cardiac actin, green) and TUNEL staining (red) of EHTs (CM+Fb+Mac) 5 days after mock or SARS-CoV-2 infection (MOI 0.1). DAPI: blue. Representative images from 4 independent experiments. b, Quantification of cell death (percent of TUNEL-positive cells) in areas of viral infection. Each data point denotes an individual EHT, bar height corresponds to the mean, error bars represent standard error of the mean, *p
    Figure Legend Snippet: Mechanisms of reduced EHT contractility. a , Combined immunostaining for cardiomyocytes (cardiac actin, green) and TUNEL staining (red) of EHTs (CM+Fb+Mac) 5 days after mock or SARS-CoV-2 infection (MOI 0.1). DAPI: blue. Representative images from 4 independent experiments. b, Quantification of cell death (percent of TUNEL-positive cells) in areas of viral infection. Each data point denotes an individual EHT, bar height corresponds to the mean, error bars represent standard error of the mean, *p

    Techniques Used: Immunostaining, TUNEL Assay, Staining, Infection

    12) Product Images from "Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues"

    Article Title: Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues

    Journal: Applied Immunohistochemistry & Molecular Morphology

    doi: 10.1097/PAI.0000000000000878

    Pellets of HEK293 cells transfected with SARS-CoV2: S1 and S2 subunit of spike protein, nucleoprotein and untransfected cells. A–D, Hematoxylin eosin stain/H  E. E–H, mAb FIPV3-20 to nucleoprotein, no immunolabeling of any pellet. I–L, mAb 019, intense immunostaining of all cell pellets. M–P, mAb 1A9, exclusive immunoreactivity of HEK293 cells transfected with spike protein S2 subunit. Q–T, mAb 001, homogeneous staining of HEK293 cells expressing nucleoprotein. U–X. In situ hybridization with probe to S1 subunit positive in corresponding HEK293 cells.
    Figure Legend Snippet: Pellets of HEK293 cells transfected with SARS-CoV2: S1 and S2 subunit of spike protein, nucleoprotein and untransfected cells. A–D, Hematoxylin eosin stain/H E. E–H, mAb FIPV3-20 to nucleoprotein, no immunolabeling of any pellet. I–L, mAb 019, intense immunostaining of all cell pellets. M–P, mAb 1A9, exclusive immunoreactivity of HEK293 cells transfected with spike protein S2 subunit. Q–T, mAb 001, homogeneous staining of HEK293 cells expressing nucleoprotein. U–X. In situ hybridization with probe to S1 subunit positive in corresponding HEK293 cells.

    Techniques Used: Transfection, Staining, Immunolabeling, Immunostaining, Expressing, In Situ Hybridization

    13) Product Images from "SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring"

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring

    Journal: Matter

    doi: 10.1016/j.matt.2020.09.027

    Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).

    Techniques Used: Construct

    Investigation of the Selectivity and Multiplexed Performance of the Wireless SARS-CoV-2 RapidPlex Platform (A) Selective response of NP, S1-IgG and S1-IgM isotypes, and CRP sensors against different non-target circulating analytes. Interferential molecules were tested at 500 pg mL −1 (with an exception of 50 ng mL −1 for CRP), 250 ng mL −1 , and 50 ng mL −1 for NP, S1-IgG and S1-IgM, and CRP assays, respectively. Data are presented as mean ± SD (n = 3). (B) Validation of sample concentrations measured using the designed electrochemical sensor against sample concentrations measured using ELISA. (C) Block diagram of the SARS-CoV-2 RapidPlex platform. UART, universal asynchronous receiver/transmitter; MCU, microcontroller unit; DAC, digital-to-analog converter; ADC, analog-to-digital converter. (D) Schematic illustration of the graphene sensor array layout. (E) Experimental readings obtained with the functionalized SARS-CoV-2 RapidPlex platform after incubation of the four WEs with PBS (pH 7.4) supplemented with 1.0% BSA containing 1.0 ng mL −1 NP (I), 250 ng mL −1 S1-IgG (II), 250 ng mL −1 S1-IgM (III), and 50 ng mL −1 CRP (IV).
    Figure Legend Snippet: Investigation of the Selectivity and Multiplexed Performance of the Wireless SARS-CoV-2 RapidPlex Platform (A) Selective response of NP, S1-IgG and S1-IgM isotypes, and CRP sensors against different non-target circulating analytes. Interferential molecules were tested at 500 pg mL −1 (with an exception of 50 ng mL −1 for CRP), 250 ng mL −1 , and 50 ng mL −1 for NP, S1-IgG and S1-IgM, and CRP assays, respectively. Data are presented as mean ± SD (n = 3). (B) Validation of sample concentrations measured using the designed electrochemical sensor against sample concentrations measured using ELISA. (C) Block diagram of the SARS-CoV-2 RapidPlex platform. UART, universal asynchronous receiver/transmitter; MCU, microcontroller unit; DAC, digital-to-analog converter; ADC, analog-to-digital converter. (D) Schematic illustration of the graphene sensor array layout. (E) Experimental readings obtained with the functionalized SARS-CoV-2 RapidPlex platform after incubation of the four WEs with PBS (pH 7.4) supplemented with 1.0% BSA containing 1.0 ng mL −1 NP (I), 250 ng mL −1 S1-IgG (II), 250 ng mL −1 S1-IgM (III), and 50 ng mL −1 CRP (IV).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation

    Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).
    Figure Legend Snippet: Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Whisker Assay

    A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS-CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.
    Figure Legend Snippet: A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS-CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.

    Techniques Used: Multiplex Assay, Biomarker Assay

    Characterization of Electrochemical Graphene Biosensors Comprising the SARS-CoV-2 RapidPlex Platform (A) Scheme detailing the methodology developed for the covalent attachment of the corresponding bioreceptor for the specific capture of the target analytes SARS-CoV-2 NP and CRP (left), and IgG and IgM isotypes against SARS-CoV-2 S1 protein (right). PBA, 1-pyrenebutyric acid; BSA, bovine serum albumin; CAb, capture antibody; PI, polyimide. (B and C) Differential pulse voltammetry (B) and Nyquist plots (C) of a graphene electrode in 0.01 M PBS (pH 7.4) containing 2.0 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1) after each modification step (S1-IgG assay as representative example): bare graphene (Bare), functionalization with PBA (PBA), immobilization of SARS-CoV-2 S1 protein (Protein), blocking with BSA (BSA), recognition of specific S1-IgG (Target), and incubation with enzyme-tagged anti-human IgG antibody (DAb). (D) Comparison of amperometric responses and overlaid signal-to-blank (S/B) ratio (black lines) for SARS-CoV-2-specific IgG and CRP detection using PBA and 1H-pyrrole-1-propionic acid (PPA) as linkers for the attachment of the corresponding capture bioreceptors. Data are presented as mean ± SD (n = 3). (E) Amperometric responses and overlaid S/B ratio (black lines) observed for 0.0 and 500 pg mL −1 NP, 0.0 and 250 ng mL −1 SARS-CoV-2-specific IgG and IgM, and 0.0 and 50 ng mL −1 CRP, with 1-, 5-, and 10-min incubation. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Characterization of Electrochemical Graphene Biosensors Comprising the SARS-CoV-2 RapidPlex Platform (A) Scheme detailing the methodology developed for the covalent attachment of the corresponding bioreceptor for the specific capture of the target analytes SARS-CoV-2 NP and CRP (left), and IgG and IgM isotypes against SARS-CoV-2 S1 protein (right). PBA, 1-pyrenebutyric acid; BSA, bovine serum albumin; CAb, capture antibody; PI, polyimide. (B and C) Differential pulse voltammetry (B) and Nyquist plots (C) of a graphene electrode in 0.01 M PBS (pH 7.4) containing 2.0 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1) after each modification step (S1-IgG assay as representative example): bare graphene (Bare), functionalization with PBA (PBA), immobilization of SARS-CoV-2 S1 protein (Protein), blocking with BSA (BSA), recognition of specific S1-IgG (Target), and incubation with enzyme-tagged anti-human IgG antibody (DAb). (D) Comparison of amperometric responses and overlaid signal-to-blank (S/B) ratio (black lines) for SARS-CoV-2-specific IgG and CRP detection using PBA and 1H-pyrrole-1-propionic acid (PPA) as linkers for the attachment of the corresponding capture bioreceptors. Data are presented as mean ± SD (n = 3). (E) Amperometric responses and overlaid S/B ratio (black lines) observed for 0.0 and 500 pg mL −1 NP, 0.0 and 250 ng mL −1 SARS-CoV-2-specific IgG and IgM, and 0.0 and 50 ng mL −1 CRP, with 1-, 5-, and 10-min incubation. Data are presented as mean ± SD (n = 3).

    Techniques Used: Modification, Blocking Assay, Incubation

    14) Product Images from "The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia"

    Article Title: The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia

    Journal: bioRxiv

    doi: 10.1101/2021.02.03.429670

    Immunofluorescence analysis of SARS-CoV-2 infection of HAE cells. HAE-ALI B2-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 or 2 pfu/cell, as indicated, mock-infected (Mock). At 4 days post-infection, a piece of the insert membrane was fixed in 4% paraformaldehyde in PBS at 4°C overnight. and subjected to direct immunofluorescence analysis. The membranes were stained with anti-SARS-CoV-2 N protein (NP). Images were taken on a Leica TCS SPE confocal microscope under 40×, which was controlled by Leica Application Suite X software. The nuclei were stained with DAPI (4’=,6-diamidino-2-phenylindole). Scale bar is 20 µM.
    Figure Legend Snippet: Immunofluorescence analysis of SARS-CoV-2 infection of HAE cells. HAE-ALI B2-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 or 2 pfu/cell, as indicated, mock-infected (Mock). At 4 days post-infection, a piece of the insert membrane was fixed in 4% paraformaldehyde in PBS at 4°C overnight. and subjected to direct immunofluorescence analysis. The membranes were stained with anti-SARS-CoV-2 N protein (NP). Images were taken on a Leica TCS SPE confocal microscope under 40×, which was controlled by Leica Application Suite X software. The nuclei were stained with DAPI (4’=,6-diamidino-2-phenylindole). Scale bar is 20 µM.

    Techniques Used: Immunofluorescence, Infection, Staining, Microscopy, Software

    Identification and quantification of SARS-CoV-2 subgenomic RNAs. (A) Genome organization. The SARS-CoV-2 genome is schematically diagrammed (not to scale) with regions in order coding for open reading frame 1a (ORF1a)/ORF1b, S protein, ORF3a, E and M proteins, ORF7a/b and ORF8, N protein, and ORF9a/b. The leader sequence was labeled as L in blue box. The structural genes are labeled within boxes in orange and the accessory genes are labeled within boxes in light green. (B) Subgenomic RNAs . Six total RNA samples were extracted from SARS-CoV-2 infected HAE-ALI cultures (at MOIs of 0.2 and 2, respectively) and subjected to whole RNA-seq. Three repeats in each MOI group were merged. Junction-spanning reads were identified using STAR (2.7.3a), and the transcript abundance, as shown in % under HAE-ALI/MOI 0.2 or 2, was estimated by counting the reads that span the junction of the corresponding RNA transcript. The left is the diagrammed subgenomic RNAs. The canonical junction-spanning reads related to each sgRNA were calculated and the ratios are shown on right. The abundances of the subgenomic transcripts identified in Vero cells in a previous study ( 15 ) are listed for comparison.
    Figure Legend Snippet: Identification and quantification of SARS-CoV-2 subgenomic RNAs. (A) Genome organization. The SARS-CoV-2 genome is schematically diagrammed (not to scale) with regions in order coding for open reading frame 1a (ORF1a)/ORF1b, S protein, ORF3a, E and M proteins, ORF7a/b and ORF8, N protein, and ORF9a/b. The leader sequence was labeled as L in blue box. The structural genes are labeled within boxes in orange and the accessory genes are labeled within boxes in light green. (B) Subgenomic RNAs . Six total RNA samples were extracted from SARS-CoV-2 infected HAE-ALI cultures (at MOIs of 0.2 and 2, respectively) and subjected to whole RNA-seq. Three repeats in each MOI group were merged. Junction-spanning reads were identified using STAR (2.7.3a), and the transcript abundance, as shown in % under HAE-ALI/MOI 0.2 or 2, was estimated by counting the reads that span the junction of the corresponding RNA transcript. The left is the diagrammed subgenomic RNAs. The canonical junction-spanning reads related to each sgRNA were calculated and the ratios are shown on right. The abundances of the subgenomic transcripts identified in Vero cells in a previous study ( 15 ) are listed for comparison.

    Techniques Used: Sequencing, Labeling, Infection, RNA Sequencing Assay

    Genome coverage of SARS-CoV-2 infected HAE cells with MOIs of 0.2 and 2, respectively. Six total RNA samples, as indicated with six colors, extracted from HAE-ALI B2-20 cultures infected with SARS-CoV-2 with at MOIs of 0.2 and 2, respectively, were subjected to whole RNA-seq. The reads were mapped to the reference SARS-CoV-2 Wuhan-Hu-1 strain genome (MN908947, NCBI), as shown with nucleotide numbers (X axis), using BWA and the sequencing read coverage (Y axis) was calculated.
    Figure Legend Snippet: Genome coverage of SARS-CoV-2 infected HAE cells with MOIs of 0.2 and 2, respectively. Six total RNA samples, as indicated with six colors, extracted from HAE-ALI B2-20 cultures infected with SARS-CoV-2 with at MOIs of 0.2 and 2, respectively, were subjected to whole RNA-seq. The reads were mapped to the reference SARS-CoV-2 Wuhan-Hu-1 strain genome (MN908947, NCBI), as shown with nucleotide numbers (X axis), using BWA and the sequencing read coverage (Y axis) was calculated.

    Techniques Used: Infection, RNA Sequencing Assay, Sequencing

    Apical virus release kinetics of SARS-CoV-2 infected HAE-ALI KC19 culture. HAE-ALI KC19 cultures were infected with SARS-CoV-2 at an MOI of 0.2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 300 µl of D-PBS to collect the released viruses. Plaque-forming units (PFU) were determined (y axis) and plotted to the dpi. Values represent means ± standard deviations (SD) (error bars).
    Figure Legend Snippet: Apical virus release kinetics of SARS-CoV-2 infected HAE-ALI KC19 culture. HAE-ALI KC19 cultures were infected with SARS-CoV-2 at an MOI of 0.2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 300 µl of D-PBS to collect the released viruses. Plaque-forming units (PFU) were determined (y axis) and plotted to the dpi. Values represent means ± standard deviations (SD) (error bars).

    Techniques Used: Infection

    Features of the S gene of SARS-CoV-2 and the deletions detected in the FCS region. (A) S gene and FCS. Key domains of the S polypeptide are diagrammed in the context of the SARS-CoV-2 genome. The S1 protein, receptor binding unit, harbors N-terminal domain (NTD) and receptor binding domain (RBD) subunit, which is conserved and recognizes ACE2. The S2, membrane fusion subunit, has fusion peptide (FP), S2’ proteolytic site, two heptad-repeats, HR1 and HR2, and a transmembrane domain (TM) followed by cytoplasmic peptide (CP) ( 30 ). The S protein has acquired a polybasic site (RRAR↓S, a furin cleavage site, FCS) for cleavage at S1/S2 boundary. An FCS region of aa670-695, together with the two key deletions mut-del1 (ΔFCS1) and mut-del2 (ΔFCS2), are shown with S amino acid sequences of the SARS-CoV-2 genome (GenBank, accession code MN908947 ). (B) Coverage plots of S gene at nt 23 , 500 to 23 , 698 in SARS-CoV-2 infected HAE-ALI B2-20 . The coverage plots show the most abundant junction-spanning reads in SARS-CoV-2 infected HAE-ALI B2-20 cultures are the 36 bp and 15 bp deletions in S gene of nt 23,594-23,629 and nt 23,583-23,597, respectively, which deleted 12 aa and 5 aa shown in mut-del1 and mut-del2 in panel A. RNA Sample 5, 6, and 7 were extracted from HAE-ALI B2-20 infected at an MOI of 0.2 at 4 dpi, and RNA Sample 9, 10, and 11 were extracted at MOI of 2 at 4 dpi.
    Figure Legend Snippet: Features of the S gene of SARS-CoV-2 and the deletions detected in the FCS region. (A) S gene and FCS. Key domains of the S polypeptide are diagrammed in the context of the SARS-CoV-2 genome. The S1 protein, receptor binding unit, harbors N-terminal domain (NTD) and receptor binding domain (RBD) subunit, which is conserved and recognizes ACE2. The S2, membrane fusion subunit, has fusion peptide (FP), S2’ proteolytic site, two heptad-repeats, HR1 and HR2, and a transmembrane domain (TM) followed by cytoplasmic peptide (CP) ( 30 ). The S protein has acquired a polybasic site (RRAR↓S, a furin cleavage site, FCS) for cleavage at S1/S2 boundary. An FCS region of aa670-695, together with the two key deletions mut-del1 (ΔFCS1) and mut-del2 (ΔFCS2), are shown with S amino acid sequences of the SARS-CoV-2 genome (GenBank, accession code MN908947 ). (B) Coverage plots of S gene at nt 23 , 500 to 23 , 698 in SARS-CoV-2 infected HAE-ALI B2-20 . The coverage plots show the most abundant junction-spanning reads in SARS-CoV-2 infected HAE-ALI B2-20 cultures are the 36 bp and 15 bp deletions in S gene of nt 23,594-23,629 and nt 23,583-23,597, respectively, which deleted 12 aa and 5 aa shown in mut-del1 and mut-del2 in panel A. RNA Sample 5, 6, and 7 were extracted from HAE-ALI B2-20 infected at an MOI of 0.2 at 4 dpi, and RNA Sample 9, 10, and 11 were extracted at MOI of 2 at 4 dpi.

    Techniques Used: Binding Assay, Infection

    15) Product Images from "Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine"

    Article Title: Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

    Journal: bioRxiv

    doi: 10.1101/2020.07.01.183236

    Humoral immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses A-B) Binding antibodies. S, RBD, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first (A) and second (B) immunization by ELISA. Dashed lines in A and B indicate median binding antibody endpoint titers measured in convalescent human sera (Figure S4). One-way ANOVA with Tukey’s multiple comparison test was used to evaluate differences between binding antibody end-point titers. C) IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). D-G) NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first (D, F) and second (E, G) immunization against SARS-CoV-2 pseudovirus (pv) (D-E) or infectious SARS-CoV-2 virus (F-G) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate (D-E) or triplicate (F-G) infection. N/A=failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. H) SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient (r) was calculated in H. *p
    Figure Legend Snippet: Humoral immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses A-B) Binding antibodies. S, RBD, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first (A) and second (B) immunization by ELISA. Dashed lines in A and B indicate median binding antibody endpoint titers measured in convalescent human sera (Figure S4). One-way ANOVA with Tukey’s multiple comparison test was used to evaluate differences between binding antibody end-point titers. C) IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). D-G) NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first (D, F) and second (E, G) immunization against SARS-CoV-2 pseudovirus (pv) (D-E) or infectious SARS-CoV-2 virus (F-G) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate (D-E) or triplicate (F-G) infection. N/A=failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. H) SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient (r) was calculated in H. *p

    Techniques Used: Mouse Assay, Recombinant, Derivative Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

    Cellular immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single or double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific cellular immune responses. Antigen-specific CD8+ ( A and B ) and CD4+ ( C and D ) T cell responses induced by the vaccine vectors after two immunizations were evaluated by flow cytometry for IFN γ , TNFα, IL-4 and IL-10 secretion following ex vivo antigen stimulation using SARS-CoV-2 S and N-specific peptide libraries. Due to technical issues, 1-3 animals/group were not included in the CD4/TNFα analysis in C and D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences in % of cytokine-specific T-cells between groups. *p
    Figure Legend Snippet: Cellular immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single or double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific cellular immune responses. Antigen-specific CD8+ ( A and B ) and CD4+ ( C and D ) T cell responses induced by the vaccine vectors after two immunizations were evaluated by flow cytometry for IFN γ , TNFα, IL-4 and IL-10 secretion following ex vivo antigen stimulation using SARS-CoV-2 S and N-specific peptide libraries. Due to technical issues, 1-3 animals/group were not included in the CD4/TNFα analysis in C and D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences in % of cytokine-specific T-cells between groups. *p

    Techniques Used: Mouse Assay, Recombinant, Derivative Assay, Flow Cytometry, Ex Vivo

    16) Product Images from "Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates"

    Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

    Journal: bioRxiv

    doi: 10.1101/2020.10.09.334136

    Protective efficacy of GX-19 against SARS-CoV-2 challenge. Non-vaccinated (n=3) and GX-19 vaccinated macaques (n=3) were challenged by intratracheal, oral, conjunctival, intranasal, and intravenous administration of 2.7 × 10 7 TCID 50 SARS-CoV-2. Viral load were assessed in nasal swab (A) and throat swab (B) at multiple time-points following challenge. Summary of peak viral loads and viral load AUC in nasal swab (C, E) and throat swab (D, F) following challenge. Dashed line indicate the assay limit of detection. Histopathological changes in the lungs of SARS-CoV-2 challenged macaques (G) . The lung tissue sections were stained with hematoxylin and eosin (H E).
    Figure Legend Snippet: Protective efficacy of GX-19 against SARS-CoV-2 challenge. Non-vaccinated (n=3) and GX-19 vaccinated macaques (n=3) were challenged by intratracheal, oral, conjunctival, intranasal, and intravenous administration of 2.7 × 10 7 TCID 50 SARS-CoV-2. Viral load were assessed in nasal swab (A) and throat swab (B) at multiple time-points following challenge. Summary of peak viral loads and viral load AUC in nasal swab (C, E) and throat swab (D, F) following challenge. Dashed line indicate the assay limit of detection. Histopathological changes in the lungs of SARS-CoV-2 challenged macaques (G) . The lung tissue sections were stained with hematoxylin and eosin (H E).

    Techniques Used: Staining

    Antibody and T-cell responses after GX-19 vaccination in macaques. Macaques (n=3) were immunized with 3 mg of GX-19 as described in the methods. Serum and PBMCs were collected before (wk 0), during (wk 4, and 5.5) and after (wk 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) and neutralizing antibodies against SARS-CoV-2 live-virus (B) . Data represent mean SEM of individual macaques, and dashed line indicate the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C) . The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D) .
    Figure Legend Snippet: Antibody and T-cell responses after GX-19 vaccination in macaques. Macaques (n=3) were immunized with 3 mg of GX-19 as described in the methods. Serum and PBMCs were collected before (wk 0), during (wk 4, and 5.5) and after (wk 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) and neutralizing antibodies against SARS-CoV-2 live-virus (B) . Data represent mean SEM of individual macaques, and dashed line indicate the assay limits of detection. The number of SARS-CoV-2 S-specific IFN-γ secreting cells in PBMCs was determined by IFN-γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells (C) . The frequency of S-specific CD4 + or CD8 + T cells producing IFN-γ, TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (D) .

    Techniques Used: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Staining

    Immunization with GX-19 elicit Th1-biased T cell responses in mice. BALB/c mice (n=3-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the methods (A-C) . Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (A) , and endpoint tier ratios of IgG2a/b to IgG1 (B) were calculated. 2 weeks post-boost mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo . Indicated cytokines in the supernatants of culture were quantified using Th1/Th2 cytometric bead array kit (D) . T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein (C) . Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (E) . Data representative of two independent experiments. P values determined by Mann-Whitney test.
    Figure Legend Snippet: Immunization with GX-19 elicit Th1-biased T cell responses in mice. BALB/c mice (n=3-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the methods (A-C) . Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (A) , and endpoint tier ratios of IgG2a/b to IgG1 (B) were calculated. 2 weeks post-boost mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo . Indicated cytokines in the supernatants of culture were quantified using Th1/Th2 cytometric bead array kit (D) . T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein (C) . Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) (E) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

    Techniques Used: Mouse Assay, Plasmid Preparation, Isolation, Ex Vivo, Enzyme-linked Immunospot, Staining, MANN-WHITNEY

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

    Techniques Used: Expressing, Mouse Assay, Plasmid Preparation

    GX-19 elicit robust binding and neutralizing antibody responses in mice. BALB/c mice (n=4-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 as described in the methods (A-C) . Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) , and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live-virus (C) . BAL were collected 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA (B) . Data representative of two independent experiments. P values determined by Mann-Whitney test.
    Figure Legend Snippet: GX-19 elicit robust binding and neutralizing antibody responses in mice. BALB/c mice (n=4-7/group) were immunized at week 0 and 2 with indicated doses of GX-19 or pGX27 as described in the methods (A-C) . Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA (A) , and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live-virus (C) . BAL were collected 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA (B) . Data representative of two independent experiments. P values determined by Mann-Whitney test.

    Techniques Used: Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    17) Product Images from "Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform"

    Article Title: Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19819-1

    Humoral immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice ( n = 5) immunized twice in a 3-week interval with 5 × 10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses. a , b Binding antibodies. S-, RBD-, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first ( a ) and second ( b ) immunization by ELISA. As a comparison, antigen-specific endpoint titers measured in n = 19 plasma samples from SARS-CoV-2 convalescent individuals (Fig. S4 ) were added in a and b . Data are presented as mean values + SD. One-way ANOVA with Tukey’s multiple comparison test was used to evaluate the differences between binding antibody end-point titers. c IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. Lines represent median values. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). d – g NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first ( d , f ) and second ( e , g ) immunization against SARS-CoV-2 pseudovirus (pv) ( d , e ) or infectious SARS-CoV-2 virus ( f , g ) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate ( d , e ) or triplicate ( f , g ) infection. N/A = failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. h SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient ( r ) was calculated in h ; *0.05
    Figure Legend Snippet: Humoral immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice ( n = 5) immunized twice in a 3-week interval with 5 × 10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses. a , b Binding antibodies. S-, RBD-, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first ( a ) and second ( b ) immunization by ELISA. As a comparison, antigen-specific endpoint titers measured in n = 19 plasma samples from SARS-CoV-2 convalescent individuals (Fig. S4 ) were added in a and b . Data are presented as mean values + SD. One-way ANOVA with Tukey’s multiple comparison test was used to evaluate the differences between binding antibody end-point titers. c IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. Lines represent median values. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). d – g NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first ( d , f ) and second ( e , g ) immunization against SARS-CoV-2 pseudovirus (pv) ( d , e ) or infectious SARS-CoV-2 virus ( f , g ) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate ( d , e ) or triplicate ( f , g ) infection. N/A = failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. h SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient ( r ) was calculated in h ; *0.05

    Techniques Used: Mouse Assay, Recombinant, Derivative Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

    Cellular immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice ( n = 5) immunized twice in a 3-week interval with 5 × 10 7 PFU of the single or double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific cellular immune responses. Antigen-specific CD8+ ( a and b ) and CD4+ ( c and d ) T-cell responses induced by the vaccine vectors after two immunizations were evaluated by flow cytometry for IFNγ, TNFα, IL-4, and IL-10 secretion following ex vivo antigen stimulation using SARS-CoV-2 S- and N-specific peptide libraries. Due to technical issues, 1–3 animals/group were not included in the CD4/TNFα analysis in c and d . One-way ANOVA with Tukey’s multiple comparison test was used to compare % of cytokine-specific T cells between immunized mice and mock controls. Lines represent median values; *0.05
    Figure Legend Snippet: Cellular immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice ( n = 5) immunized twice in a 3-week interval with 5 × 10 7 PFU of the single or double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific cellular immune responses. Antigen-specific CD8+ ( a and b ) and CD4+ ( c and d ) T-cell responses induced by the vaccine vectors after two immunizations were evaluated by flow cytometry for IFNγ, TNFα, IL-4, and IL-10 secretion following ex vivo antigen stimulation using SARS-CoV-2 S- and N-specific peptide libraries. Due to technical issues, 1–3 animals/group were not included in the CD4/TNFα analysis in c and d . One-way ANOVA with Tukey’s multiple comparison test was used to compare % of cytokine-specific T cells between immunized mice and mock controls. Lines represent median values; *0.05

    Techniques Used: Mouse Assay, Recombinant, Derivative Assay, Flow Cytometry, Ex Vivo

    CD8+ T-cell responses induced by sMVA-N/S in HLA transgenic mice. HLA-B*07:02 (B7) transgenic mice ( n = 4) were immunized twice in a 3-week interval with 1 × 10 7 PFU of sMVA-N/S or sMVA control vector (reconstituted with FPV TROVAC). B7 mice ( n = 3) were mock-immunized as additional control. Development of SARS-CoV-2-specific CD8+ T cells was evaluated 1 week post booster immunization. a – d Intracellular cytokine staining. Nucleocapsid- ( a , c ) and Spike-specific ( b , d ) CD8+ T cells were evaluated by intracellular cytokine staining for IFNγ, TNFα, and IL-4 secretion following ex vivo antigen stimulation by N and S peptide libraries, respectively. Panels a and b show the percentage of CD3+/CD8+ T cells secreting IFNγ, TNFα, or IL-4 following peptide stimulation. Panels c and d show relative frequencies of CD8+ T cells secreting one or more cytokines after peptide stimulation. Total percentage of cytokine-secreting cells within CD3+/CD8+ population is indicated under each pie chart. e ELISPOT analysis of IFNγ-secreting cells following stimulation with S and N peptide libraries, S library sub-pools (1S1, 2S1, S2), and N26 peptide containing the HLA-B*07:02-restricted N-specific immunodominant epitope SPRWYFYYL. One-way ANOVA with Dunnett’s multiple comparison test was used in a and b . Two-way ANOVA with Dunnett’s multiple comparison test was used in e . Data in a , b , and e are presented as mean values ± SD; *0.05
    Figure Legend Snippet: CD8+ T-cell responses induced by sMVA-N/S in HLA transgenic mice. HLA-B*07:02 (B7) transgenic mice ( n = 4) were immunized twice in a 3-week interval with 1 × 10 7 PFU of sMVA-N/S or sMVA control vector (reconstituted with FPV TROVAC). B7 mice ( n = 3) were mock-immunized as additional control. Development of SARS-CoV-2-specific CD8+ T cells was evaluated 1 week post booster immunization. a – d Intracellular cytokine staining. Nucleocapsid- ( a , c ) and Spike-specific ( b , d ) CD8+ T cells were evaluated by intracellular cytokine staining for IFNγ, TNFα, and IL-4 secretion following ex vivo antigen stimulation by N and S peptide libraries, respectively. Panels a and b show the percentage of CD3+/CD8+ T cells secreting IFNγ, TNFα, or IL-4 following peptide stimulation. Panels c and d show relative frequencies of CD8+ T cells secreting one or more cytokines after peptide stimulation. Total percentage of cytokine-secreting cells within CD3+/CD8+ population is indicated under each pie chart. e ELISPOT analysis of IFNγ-secreting cells following stimulation with S and N peptide libraries, S library sub-pools (1S1, 2S1, S2), and N26 peptide containing the HLA-B*07:02-restricted N-specific immunodominant epitope SPRWYFYYL. One-way ANOVA with Dunnett’s multiple comparison test was used in a and b . Two-way ANOVA with Dunnett’s multiple comparison test was used in e . Data in a , b , and e are presented as mean values ± SD; *0.05

    Techniques Used: Transgenic Assay, Mouse Assay, Plasmid Preparation, Staining, Ex Vivo, Enzyme-linked Immunospot

    18) Product Images from "SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring"

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring

    Journal: Matter

    doi: 10.1016/j.matt.2020.09.027

    Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Evaluation of Analytical Sensor Performance for the Detection of Physiological Levels of Target COVID-19 Biomarkers (A) Scheme of sensor preparation for detection of SARS-CoV-2 NP and CRP based on double-sandwich and sandwich assay configurations, respectively. CAb, capture antibody; DAb, detector antibody; DAb 2 , secondary detector antibody; HRP, horseradish peroxidase. (B and C) Calibration curves constructed for NP (B) and CRP (C) detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3). (D) Scheme of sensor preparation for detection of S1-IgG and S1-IgM isotypes based on direct assay configurations. (E and F) Calibration curves constructed for S1-IgG (E) and S1-IgM (F) isotype detection in PBS (pH 7.4) supplemented with 1.0% BSA. Data are presented as mean ± SD (n = 3).

    Techniques Used: Construct

    Investigation of the Selectivity and Multiplexed Performance of the Wireless SARS-CoV-2 RapidPlex Platform (A) Selective response of NP, S1-IgG and S1-IgM isotypes, and CRP sensors against different non-target circulating analytes. Interferential molecules were tested at 500 pg mL −1 (with an exception of 50 ng mL −1 for CRP), 250 ng mL −1 , and 50 ng mL −1 for NP, S1-IgG and S1-IgM, and CRP assays, respectively. Data are presented as mean ± SD (n = 3). (B) Validation of sample concentrations measured using the designed electrochemical sensor against sample concentrations measured using ELISA. (C) Block diagram of the SARS-CoV-2 RapidPlex platform. UART, universal asynchronous receiver/transmitter; MCU, microcontroller unit; DAC, digital-to-analog converter; ADC, analog-to-digital converter. (D) Schematic illustration of the graphene sensor array layout. (E) Experimental readings obtained with the functionalized SARS-CoV-2 RapidPlex platform after incubation of the four WEs with PBS (pH 7.4) supplemented with 1.0% BSA containing 1.0 ng mL −1 NP (I), 250 ng mL −1 S1-IgG (II), 250 ng mL −1 S1-IgM (III), and 50 ng mL −1 CRP (IV).
    Figure Legend Snippet: Investigation of the Selectivity and Multiplexed Performance of the Wireless SARS-CoV-2 RapidPlex Platform (A) Selective response of NP, S1-IgG and S1-IgM isotypes, and CRP sensors against different non-target circulating analytes. Interferential molecules were tested at 500 pg mL −1 (with an exception of 50 ng mL −1 for CRP), 250 ng mL −1 , and 50 ng mL −1 for NP, S1-IgG and S1-IgM, and CRP assays, respectively. Data are presented as mean ± SD (n = 3). (B) Validation of sample concentrations measured using the designed electrochemical sensor against sample concentrations measured using ELISA. (C) Block diagram of the SARS-CoV-2 RapidPlex platform. UART, universal asynchronous receiver/transmitter; MCU, microcontroller unit; DAC, digital-to-analog converter; ADC, analog-to-digital converter. (D) Schematic illustration of the graphene sensor array layout. (E) Experimental readings obtained with the functionalized SARS-CoV-2 RapidPlex platform after incubation of the four WEs with PBS (pH 7.4) supplemented with 1.0% BSA containing 1.0 ng mL −1 NP (I), 250 ng mL −1 S1-IgG (II), 250 ng mL −1 S1-IgM (III), and 50 ng mL −1 CRP (IV).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation

    Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).
    Figure Legend Snippet: Application of SARS-CoV-2 RapidPlex in SARS-CoV-2 Detection in Blood and Saliva Samples from COVID-19-Positive and -Negative Subjects (A and B) Experimental readings obtained with SARS-CoV-2 RapidPlex after 10-min incubation of the sensor array with serum samples from a representative COVID-19 RT-PCR-negative (A) and -positive (B) patient. (C) Signal of individual sensor obtained after 1-min incubation with a serum sample from a COVID-19-positive patient (dark color) versus the signal obtained after 10-min incubation with a serum sample from a COVID-19-negative patient (light color). (D) Box-and-whisker plot of measured signal-to-blank ratios (S/B) for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 6) serum samples. (E) Box-and-whisker plot of measured S/B for NP, S1-IgG, S1-IgM, and CRP in RT-PCR-confirmed COVID-19-positive (n = 5) and -negative (n = 3) saliva samples. (F) CRP levels in diluted serum samples plotted against given COVID-19 symptom severity, with “Healthy” referring to COVID-19-negative patient samples (n = 7). Positive COVID-19 patients were classified according to disease severity as asymptomatic (n = 2), mild (n = 5), and moderate (n = 2).

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Whisker Assay

    A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS-CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.
    Figure Legend Snippet: A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS-CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.

    Techniques Used: Multiplex Assay, Biomarker Assay

    Characterization of Electrochemical Graphene Biosensors Comprising the SARS-CoV-2 RapidPlex Platform (A) Scheme detailing the methodology developed for the covalent attachment of the corresponding bioreceptor for the specific capture of the target analytes SARS-CoV-2 NP and CRP (left), and IgG and IgM isotypes against SARS-CoV-2 S1 protein (right). PBA, 1-pyrenebutyric acid; BSA, bovine serum albumin; CAb, capture antibody; PI, polyimide. (B and C) Differential pulse voltammetry (B) and Nyquist plots (C) of a graphene electrode in 0.01 M PBS (pH 7.4) containing 2.0 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1) after each modification step (S1-IgG assay as representative example): bare graphene (Bare), functionalization with PBA (PBA), immobilization of SARS-CoV-2 S1 protein (Protein), blocking with BSA (BSA), recognition of specific S1-IgG (Target), and incubation with enzyme-tagged anti-human IgG antibody (DAb). (D) Comparison of amperometric responses and overlaid signal-to-blank (S/B) ratio (black lines) for SARS-CoV-2-specific IgG and CRP detection using PBA and 1H-pyrrole-1-propionic acid (PPA) as linkers for the attachment of the corresponding capture bioreceptors. Data are presented as mean ± SD (n = 3). (E) Amperometric responses and overlaid S/B ratio (black lines) observed for 0.0 and 500 pg mL −1 NP, 0.0 and 250 ng mL −1 SARS-CoV-2-specific IgG and IgM, and 0.0 and 50 ng mL −1 CRP, with 1-, 5-, and 10-min incubation. Data are presented as mean ± SD (n = 3).
    Figure Legend Snippet: Characterization of Electrochemical Graphene Biosensors Comprising the SARS-CoV-2 RapidPlex Platform (A) Scheme detailing the methodology developed for the covalent attachment of the corresponding bioreceptor for the specific capture of the target analytes SARS-CoV-2 NP and CRP (left), and IgG and IgM isotypes against SARS-CoV-2 S1 protein (right). PBA, 1-pyrenebutyric acid; BSA, bovine serum albumin; CAb, capture antibody; PI, polyimide. (B and C) Differential pulse voltammetry (B) and Nyquist plots (C) of a graphene electrode in 0.01 M PBS (pH 7.4) containing 2.0 mM K 4 Fe(CN) 6 /K 3 Fe(CN) 6 (1:1) after each modification step (S1-IgG assay as representative example): bare graphene (Bare), functionalization with PBA (PBA), immobilization of SARS-CoV-2 S1 protein (Protein), blocking with BSA (BSA), recognition of specific S1-IgG (Target), and incubation with enzyme-tagged anti-human IgG antibody (DAb). (D) Comparison of amperometric responses and overlaid signal-to-blank (S/B) ratio (black lines) for SARS-CoV-2-specific IgG and CRP detection using PBA and 1H-pyrrole-1-propionic acid (PPA) as linkers for the attachment of the corresponding capture bioreceptors. Data are presented as mean ± SD (n = 3). (E) Amperometric responses and overlaid S/B ratio (black lines) observed for 0.0 and 500 pg mL −1 NP, 0.0 and 250 ng mL −1 SARS-CoV-2-specific IgG and IgM, and 0.0 and 50 ng mL −1 CRP, with 1-, 5-, and 10-min incubation. Data are presented as mean ± SD (n = 3).

    Techniques Used: Modification, Blocking Assay, Incubation

    19) Product Images from "Neuropathology of patients with COVID-19 in Germany: a post-mortem case series"

    Article Title: Neuropathology of patients with COVID-19 in Germany: a post-mortem case series

    Journal: The Lancet. Neurology

    doi: 10.1016/S1474-4422(20)30308-2

    Neuropathological findings and SARS-CoV-2 viral loads in studied patients (n=43) Cases are arranged from left to right on the basis of the presence and quantity of SARS-CoV-2 in the brain. F=female. FFPE=formalin-fixed paraffin-embedded. HPF=high-power field. IHC=immunohistochemistry. M=male. P=parenchymal. PV=perivascular. qPCR=quantitative PCR. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. *Values shown for positive cases represent number of copies of SARS-CoV-2 RNA (× 10 3 /mL); detection was done in the frontal lobe in cryopreserved specimens and in the upper medulla oblongata in FFPE specimens.
    Figure Legend Snippet: Neuropathological findings and SARS-CoV-2 viral loads in studied patients (n=43) Cases are arranged from left to right on the basis of the presence and quantity of SARS-CoV-2 in the brain. F=female. FFPE=formalin-fixed paraffin-embedded. HPF=high-power field. IHC=immunohistochemistry. M=male. P=parenchymal. PV=perivascular. qPCR=quantitative PCR. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. *Values shown for positive cases represent number of copies of SARS-CoV-2 RNA (× 10 3 /mL); detection was done in the frontal lobe in cryopreserved specimens and in the upper medulla oblongata in FFPE specimens.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Real-time Polymerase Chain Reaction

    Distribution of SARS-CoV-2 within the CNS Representative images of viral protein-positive cells (green arrows) in the medulla oblongata detected by anti-nucleocapsid protein antibody (A) or anti-spike protein antibody (B). (C) SARS-CoV-2 nucleoprotein (brown staining ) could also be detected in subsets of cranial nerves originating from the lower brainstem. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Distribution of SARS-CoV-2 within the CNS Representative images of viral protein-positive cells (green arrows) in the medulla oblongata detected by anti-nucleocapsid protein antibody (A) or anti-spike protein antibody (B). (C) SARS-CoV-2 nucleoprotein (brown staining ) could also be detected in subsets of cranial nerves originating from the lower brainstem. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.

    Techniques Used: Staining

    Related Articles

    High Performance Liquid Chromatography:

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring
    Article Snippet: .. Mouse NP monoclonal antibody (mAb) (40143-MM05), SARS-CoV-2 NP antigen (40588-V08B), SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit mAb (40143-R001), SARS-CoV NP antigen (HCoV-OC43; 40643-V07E), SARS-CoV-2 Spike S1-His recombinant protein (HPLC-verified) (40591-V08H), and SARS-CoV Spike S1 protein (S1 subunit, His tag) (40150-V08B1) were purchased from Sino Biological. ..

    Immunohistochemistry:

    Article Title: Neuropathology of patients with COVID-19 in Germany: a post-mortem case series
    Article Snippet: .. In specimens with sufficient high-quality tissue, we tested for the presence of the virus with immunohistochemistry using antibodies against viral nucleocapsid protein (catalogue numbers 40143-R001 [dilution 1:5000] and 40143-T62 [dilution 1:1000]; Sino Biological, Eschborn, Germany) and spike protein (clone 1A9, catalogue number GTX632604; GeneTex, Irvine, USA; dilution 1:300). .. Immunohistochemical staining was done with a Ventana Benchmark XT Autostainer.

    Blocking Assay:

    Article Title: Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro
    Article Snippet: .. The fixed plates were washed three times with 1 × PBS containing 0.05% Tween 20 (wash buffer) and blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween 20 in 1 × PBS for 1 h. After washing the plates thrice with wash buffer, the SARS-CoV/SARS-CoV-2 nucleocapsid mAb (40143-R001; Sino Biological, United States) with a dilution of 1:5,000 in 1 × PBS containing 0.5% BSA and 0.1% Tween 20 was added to each well and incubated for 2 h at 37°C. .. The plates were then washed three times with wash buffer, and HRP-conjugated goat anti-rabbit polyclonal antibody (P0448; Dako, Denmark) was used as secondary antibody at a dilution 1:2,000.

    Incubation:

    Article Title: Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro
    Article Snippet: .. The fixed plates were washed three times with 1 × PBS containing 0.05% Tween 20 (wash buffer) and blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween 20 in 1 × PBS for 1 h. After washing the plates thrice with wash buffer, the SARS-CoV/SARS-CoV-2 nucleocapsid mAb (40143-R001; Sino Biological, United States) with a dilution of 1:5,000 in 1 × PBS containing 0.5% BSA and 0.1% Tween 20 was added to each well and incubated for 2 h at 37°C. .. The plates were then washed three times with wash buffer, and HRP-conjugated goat anti-rabbit polyclonal antibody (P0448; Dako, Denmark) was used as secondary antibody at a dilution 1:2,000.

    Article Title: The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia
    Article Snippet: .. Following permeabilization with 0.2% Triton X-100 for 15 min at room temperature, the slide was incubated with a rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (# 40143-R001; SinoBiological US, Wayne, PA) at a dilution of 1:25 in PBS with 2% fetal bovine serum for 1 h at 37°C. .. After washing, the slide was incubated with a rhodamine-conjugated secondary antibody, followed by staining of the nuclei with DAPI (4’,6-diamidino-2-phenylindole).

    other:

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium
    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Article Title: Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium
    Article Snippet: Antibodies used.Primary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (clone 001) (catalog no. 40143-R001; SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6) (catalog no. T7941; MilliporeSigma, St. Louis, MO) at 1:100, mouse anti-ZO-1 (clone 1/ZO-1) (catalog no. 610966; BD Bioscience, San Jose, CA) at 1:100, and rabbit anti-Ki67 (clone SP6) (ab1666; Abcam, Cambridge, MA) at 1:50.

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: 40143-R001 (capture) and 40143-MM05 (detection) were used for N detection.

    Recombinant:

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring
    Article Snippet: .. Mouse NP monoclonal antibody (mAb) (40143-MM05), SARS-CoV-2 NP antigen (40588-V08B), SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit mAb (40143-R001), SARS-CoV NP antigen (HCoV-OC43; 40643-V07E), SARS-CoV-2 Spike S1-His recombinant protein (HPLC-verified) (40591-V08H), and SARS-CoV Spike S1 protein (S1 subunit, His tag) (40150-V08B1) were purchased from Sino Biological. ..

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    Sino Biological sars cov sars cov 2 nucleocapsid antibody rabbit mab
    Binding of plant-produced monoclonal antibodies (mAbs) B38 and H4 to severe acute respiratory syndrome coronavirus 2 <t>(SARS-CoV-2)</t> receptor binding domain (RBD) protein. The ability of the plant-produced mAbs to recognize RBD protein of SARS-CoV-2 was assessed by ELISA. The plant-produced mAbs B38 and H4, standard human immunoglobulin G (IgG)1, and plant-produced anti- PD1 antibody (as negative control) ( Rattanapisit et al., 2019b ) were incubated on plates coated with commercial SARS-CoV-2 RBD. The bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-human kappa antibody. The data are the mean values of technical triplicates per concentration.
    Sars Cov Sars Cov 2 Nucleocapsid Antibody Rabbit Mab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Binding of plant-produced monoclonal antibodies (mAbs) B38 and H4 to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) protein. The ability of the plant-produced mAbs to recognize RBD protein of SARS-CoV-2 was assessed by ELISA. The plant-produced mAbs B38 and H4, standard human immunoglobulin G (IgG)1, and plant-produced anti- PD1 antibody (as negative control) ( Rattanapisit et al., 2019b ) were incubated on plates coated with commercial SARS-CoV-2 RBD. The bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-human kappa antibody. The data are the mean values of technical triplicates per concentration.

    Journal: Frontiers in Plant Science

    Article Title: Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

    doi: 10.3389/fpls.2020.589995

    Figure Lengend Snippet: Binding of plant-produced monoclonal antibodies (mAbs) B38 and H4 to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) protein. The ability of the plant-produced mAbs to recognize RBD protein of SARS-CoV-2 was assessed by ELISA. The plant-produced mAbs B38 and H4, standard human immunoglobulin G (IgG)1, and plant-produced anti- PD1 antibody (as negative control) ( Rattanapisit et al., 2019b ) were incubated on plates coated with commercial SARS-CoV-2 RBD. The bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-human kappa antibody. The data are the mean values of technical triplicates per concentration.

    Article Snippet: The fixed plates were washed three times with 1 × PBS containing 0.05% Tween 20 (wash buffer) and blocked with blocking buffer containing 2% bovine serum albumin (BSA) and 0.1% tween 20 in 1 × PBS for 1 h. After washing the plates thrice with wash buffer, the SARS-CoV/SARS-CoV-2 nucleocapsid mAb (40143-R001; Sino Biological, United States) with a dilution of 1:5,000 in 1 × PBS containing 0.5% BSA and 0.1% Tween 20 was added to each well and incubated for 2 h at 37°C.

    Techniques: Binding Assay, Produced, Enzyme-linked Immunosorbent Assay, Negative Control, Incubation, Concentration Assay

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

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

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

    Article Snippet: 40143-R001 (capture) and 40143-MM05 (detection) were used for N detection.

    Techniques: Enzyme-linked Immunosorbent Assay

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

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

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

    Article Snippet: 40143-R001 (capture) and 40143-MM05 (detection) were used for N detection.

    Techniques: Standard Deviation

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

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

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

    Article Snippet: 40143-R001 (capture) and 40143-MM05 (detection) were used for N detection.

    Techniques: 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.

    Journal: Biosensors & Bioelectronics

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

    doi: 10.1016/j.bios.2020.112572

    Figure Lengend Snippet: 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.

    Article Snippet: 40143-R001 (capture) and 40143-MM05 (detection) were used for N detection.

    Techniques: Chemiluminescent ELISA

    SARS-CoV-2 Infects Astrocytes in Cortical Organoids A) Viral infection of cortical organoids. Human stem cells from several lines were aggregated and differentiated toward cortical identity in suspension. After 5, 10, 16 or 22 weeks of differentiation, organoids were exposed to SARS-CoV-2 for 2 hours, media was replaced and then collected 72 hours later. B) After 5, 10, or 16 weeks organoids only indicated rare infection (white arrowheads). At five and 10 weeks, SARS-CoV-2 N+ cells did not co-express SOX2, NEUN or GFAP indicating infected cells are not cortical progenitors, neurons or astrocytes and may instead be an off-target population. However, after 16 weeks, in one stem cell line a few GFAP+ astrocytes were infected. C) Although rare cells are infected at neurogenic stages, as indicated by coronavirus N antibody presence, there was no observed viral replication with dsRNA at these timepoints. D) At late gliogenic stages, by week 22 infection was readily observed in GFAP astrocytes but not NEUN+ neurons. E) 94% of infected cells stained positive for markers of astrocytes GFAP or AQP4, while only 4% are NEUN+ neurons. White arrowheads indicate SARS-CoV-2+ dsRNA+ GFAP+ AQP4+ astrocytes (GFAP+AQP4+ n=169 cells, NEUN n=143 cells).

    Journal: bioRxiv

    Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

    doi: 10.1101/2021.01.17.427024

    Figure Lengend Snippet: SARS-CoV-2 Infects Astrocytes in Cortical Organoids A) Viral infection of cortical organoids. Human stem cells from several lines were aggregated and differentiated toward cortical identity in suspension. After 5, 10, 16 or 22 weeks of differentiation, organoids were exposed to SARS-CoV-2 for 2 hours, media was replaced and then collected 72 hours later. B) After 5, 10, or 16 weeks organoids only indicated rare infection (white arrowheads). At five and 10 weeks, SARS-CoV-2 N+ cells did not co-express SOX2, NEUN or GFAP indicating infected cells are not cortical progenitors, neurons or astrocytes and may instead be an off-target population. However, after 16 weeks, in one stem cell line a few GFAP+ astrocytes were infected. C) Although rare cells are infected at neurogenic stages, as indicated by coronavirus N antibody presence, there was no observed viral replication with dsRNA at these timepoints. D) At late gliogenic stages, by week 22 infection was readily observed in GFAP astrocytes but not NEUN+ neurons. E) 94% of infected cells stained positive for markers of astrocytes GFAP or AQP4, while only 4% are NEUN+ neurons. White arrowheads indicate SARS-CoV-2+ dsRNA+ GFAP+ AQP4+ astrocytes (GFAP+AQP4+ n=169 cells, NEUN n=143 cells).

    Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061-2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419-1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735-1-AP, 1:100), Aqp4 (Proteintech, 16473-1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940-1-AP, 1:50), CD147 (Invitrogen, 34-5600, 1:500), Arcn1 (Proteintech, 23843-1-AP, 1:50), Rat: Gfap (Thermofisher, 13-0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R & D, AF933, 1:200), Ace2 (Thermofisher, MA5-32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R & D, AF385, 1:100), Sheep: Eomes (R & D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R & D, AF7227, 1:200).

    Techniques: Infection, Staining

    Coronavirus Protease Expression in Developing Human Cortex A) Single-cell RNA sequencing data from the developing cortex demonstrates minimal expression of canonical SARS-CoV-2 proteases TMPRSS2 and TMPRSS4 in cortical cell types. B) Alternative coronavirus proteases, TMPRSS5, FURIN and CTSB, are differentially expressed in a variety of cell types in the developing human cortex.

    Journal: bioRxiv

    Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

    doi: 10.1101/2021.01.17.427024

    Figure Lengend Snippet: Coronavirus Protease Expression in Developing Human Cortex A) Single-cell RNA sequencing data from the developing cortex demonstrates minimal expression of canonical SARS-CoV-2 proteases TMPRSS2 and TMPRSS4 in cortical cell types. B) Alternative coronavirus proteases, TMPRSS5, FURIN and CTSB, are differentially expressed in a variety of cell types in the developing human cortex.

    Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061-2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419-1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735-1-AP, 1:100), Aqp4 (Proteintech, 16473-1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940-1-AP, 1:50), CD147 (Invitrogen, 34-5600, 1:500), Arcn1 (Proteintech, 23843-1-AP, 1:50), Rat: Gfap (Thermofisher, 13-0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R & D, AF933, 1:200), Ace2 (Thermofisher, MA5-32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R & D, AF385, 1:100), Sheep: Eomes (R & D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R & D, AF7227, 1:200).

    Techniques: Expressing, RNA Sequencing Assay

    SARS-CoV-2 modestly infects other neural cell types A) Mature and precursor astroglial cells indicate high infection where 74% express PAX6, 56% express HOPX and 44% express S100B (PAX6 n=252 cells, HOPX n=405 cells, S100B n=343 across two biological samples and four technical replicates). B) Oligodendrocyte Precursor Cells (OPC) and Microglia have little expression compared to astrocytes (OLIG2 n=487 cells, IBA1 n=350 cells). C) Neurogenic intermediate progenitor cells (IPCs) also demonstrates minimal infection of excitatory neuron lineage (EOMES n=128 cells).

    Journal: bioRxiv

    Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

    doi: 10.1101/2021.01.17.427024

    Figure Lengend Snippet: SARS-CoV-2 modestly infects other neural cell types A) Mature and precursor astroglial cells indicate high infection where 74% express PAX6, 56% express HOPX and 44% express S100B (PAX6 n=252 cells, HOPX n=405 cells, S100B n=343 across two biological samples and four technical replicates). B) Oligodendrocyte Precursor Cells (OPC) and Microglia have little expression compared to astrocytes (OLIG2 n=487 cells, IBA1 n=350 cells). C) Neurogenic intermediate progenitor cells (IPCs) also demonstrates minimal infection of excitatory neuron lineage (EOMES n=128 cells).

    Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061-2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419-1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735-1-AP, 1:100), Aqp4 (Proteintech, 16473-1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940-1-AP, 1:50), CD147 (Invitrogen, 34-5600, 1:500), Arcn1 (Proteintech, 23843-1-AP, 1:50), Rat: Gfap (Thermofisher, 13-0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R & D, AF933, 1:200), Ace2 (Thermofisher, MA5-32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R & D, AF385, 1:100), Sheep: Eomes (R & D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R & D, AF7227, 1:200).

    Techniques: Infection, Expressing

    SARS-CoV-2 Infection Increases Cell Stress and Reactivity in Cortical Astrocytes A) After SAR-CoV-2 infection, there is no immediate increase in cell death in infected cells, as indicated by Cleaved Caspase 3 staining. B) Infected cells have an increase in cell stress, as indicated by the ER stress marker, ARCN1. C) Approximately one-third of infected astrocytes in primary cortical tissue express the reactive marker SYNM and three-quarters have the receptor for growth factor signaling, EGFR (SYNM n=62 cells, EGFR n=142 cells). D) The same proportion of infected organoid cells express SYNM and about one-half express EGFR (SYNM n=172 cells, EGFR n=143 cells).

    Journal: bioRxiv

    Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

    doi: 10.1101/2021.01.17.427024

    Figure Lengend Snippet: SARS-CoV-2 Infection Increases Cell Stress and Reactivity in Cortical Astrocytes A) After SAR-CoV-2 infection, there is no immediate increase in cell death in infected cells, as indicated by Cleaved Caspase 3 staining. B) Infected cells have an increase in cell stress, as indicated by the ER stress marker, ARCN1. C) Approximately one-third of infected astrocytes in primary cortical tissue express the reactive marker SYNM and three-quarters have the receptor for growth factor signaling, EGFR (SYNM n=62 cells, EGFR n=142 cells). D) The same proportion of infected organoid cells express SYNM and about one-half express EGFR (SYNM n=172 cells, EGFR n=143 cells).

    Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061-2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419-1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735-1-AP, 1:100), Aqp4 (Proteintech, 16473-1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940-1-AP, 1:50), CD147 (Invitrogen, 34-5600, 1:500), Arcn1 (Proteintech, 23843-1-AP, 1:50), Rat: Gfap (Thermofisher, 13-0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R & D, AF933, 1:200), Ace2 (Thermofisher, MA5-32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R & D, AF385, 1:100), Sheep: Eomes (R & D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R & D, AF7227, 1:200).

    Techniques: Infection, Staining, Marker

    Coronavirus Receptors, DPP4 and CD147, but not ACE2 are Expressed in Developing Human Cortex A) ACE2 expressing cells are readily infected by SARS-CoV-2, where they have abundant ACE2 and dsRNA presence. Primary cortical tissue and cortical organoids do not have observable ACE2 protein in cortical tissue or infected cells. NRP1 is present in cortical neurons, but not in infected cells. B) Infected cortical astrocytes in the SVZ of primary cortex abundantly express coronavirus receptors DPP4 and CD147, where 100% of cells assayed have DPP4 and 73% have CD147. In cortical organoids, 60% are DPP4+ and 28% are CD147+ (Primary: DPP4 n=61cells, CD147 n=83 cells; Organoid: DPP4 n=296 cells, CD147 n=239 cells) C) Inhibition of DPP4 by Vildagliptin results in a 30% decrease in the number of SARS-CoV-2 N+ cells and a 70% reduction in dsRNA+ cells. SARS-CoV-2 N+ dsRNA+ cells are indicated by white arrowheads (SARS-CoV-2 MOI 0.5 n=1273 cells, MOI 0.5+Vildagliptin n=879 cells, dsRNA MOI 0.5 n=571 cells, MOI 0.5+Vildagliptin n=227 cells across two biological samples and three technical replicates). D) ARCN1 is broadly expressed in SARS-CoV-2 infected samples, which is reduced by 70% after DPP4 inhibition by Vildagliptin. ARCN1+ dsRNA+ cells indicated by white arrowheads (MOI 0.5 n=1224 cells, MOI 0.5+ Vildagliptin n=331 cells across two biological samples and three technical replicates).

    Journal: bioRxiv

    Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

    doi: 10.1101/2021.01.17.427024

    Figure Lengend Snippet: Coronavirus Receptors, DPP4 and CD147, but not ACE2 are Expressed in Developing Human Cortex A) ACE2 expressing cells are readily infected by SARS-CoV-2, where they have abundant ACE2 and dsRNA presence. Primary cortical tissue and cortical organoids do not have observable ACE2 protein in cortical tissue or infected cells. NRP1 is present in cortical neurons, but not in infected cells. B) Infected cortical astrocytes in the SVZ of primary cortex abundantly express coronavirus receptors DPP4 and CD147, where 100% of cells assayed have DPP4 and 73% have CD147. In cortical organoids, 60% are DPP4+ and 28% are CD147+ (Primary: DPP4 n=61cells, CD147 n=83 cells; Organoid: DPP4 n=296 cells, CD147 n=239 cells) C) Inhibition of DPP4 by Vildagliptin results in a 30% decrease in the number of SARS-CoV-2 N+ cells and a 70% reduction in dsRNA+ cells. SARS-CoV-2 N+ dsRNA+ cells are indicated by white arrowheads (SARS-CoV-2 MOI 0.5 n=1273 cells, MOI 0.5+Vildagliptin n=879 cells, dsRNA MOI 0.5 n=571 cells, MOI 0.5+Vildagliptin n=227 cells across two biological samples and three technical replicates). D) ARCN1 is broadly expressed in SARS-CoV-2 infected samples, which is reduced by 70% after DPP4 inhibition by Vildagliptin. ARCN1+ dsRNA+ cells indicated by white arrowheads (MOI 0.5 n=1224 cells, MOI 0.5+ Vildagliptin n=331 cells across two biological samples and three technical replicates).

    Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061-2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419-1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735-1-AP, 1:100), Aqp4 (Proteintech, 16473-1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940-1-AP, 1:50), CD147 (Invitrogen, 34-5600, 1:500), Arcn1 (Proteintech, 23843-1-AP, 1:50), Rat: Gfap (Thermofisher, 13-0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R & D, AF933, 1:200), Ace2 (Thermofisher, MA5-32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R & D, AF385, 1:100), Sheep: Eomes (R & D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R & D, AF7227, 1:200).

    Techniques: Expressing, Infection, Inhibition

    A diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. ( A ) HAE-ALI model: Epithelial cells are taken from bronchia of the lung of healthy donors and plated onto Transwell ® inserts at an air-liquid interface (ALI) for four weeks. Four major types of the epithelial cells in the well differentiated polarized HAE cultures: basal, ciliated, goblet, and club cells are diagrammed in the Transwell ® insert, and their expression makers are indicated. ( B ) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CYKT5. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue). ( C ) Model of airway cell regeneration model of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, in which it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: A diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. ( A ) HAE-ALI model: Epithelial cells are taken from bronchia of the lung of healthy donors and plated onto Transwell ® inserts at an air-liquid interface (ALI) for four weeks. Four major types of the epithelial cells in the well differentiated polarized HAE cultures: basal, ciliated, goblet, and club cells are diagrammed in the Transwell ® insert, and their expression makers are indicated. ( B ) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CYKT5. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue). ( C ) Model of airway cell regeneration model of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, in which it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Expressing, Staining

    Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE-ALI over a course of 21 days. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi). Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE-ALI over a course of 21 days. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi). Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Immunofluorescence, Infection, Staining

    SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV ( A ), and anti-MUC5AC ( B ), anti-cytokeratin 5 ( C ), and anti-SCGB1A1 ( D ), respectively. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV ( A ), and anti-MUC5AC ( B ), anti-cytokeratin 5 ( C ), and anti-SCGB1A1 ( D ), respectively. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Staining

    Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Immunofluorescence, Infection, Staining

    Three-dimensional (z-stack) imaging of SARS-CoV-2 infected primary bronchial HAE-ALI. Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or with anti-NP and anti-β-tubulin IV antibodies (B), or co-stained anti-NP and anti-ZO-1 antibodies ( B ). A set of confocal images were taken at a magnification of x 40 from the stained pierce of epithelium at a distance of the Z value (μm), shown in each image, from the objective (z-axis) and reconstituted as a 3-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Three-dimensional (z-stack) imaging of SARS-CoV-2 infected primary bronchial HAE-ALI. Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or with anti-NP and anti-β-tubulin IV antibodies (B), or co-stained anti-NP and anti-ZO-1 antibodies ( B ). A set of confocal images were taken at a magnification of x 40 from the stained pierce of epithelium at a distance of the Z value (μm), shown in each image, from the objective (z-axis) and reconstituted as a 3-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Imaging, Infection, Staining, Fluorescence

    Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection). (A C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A), 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days post-infection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque forming units (pfu) were plotted to the DPI. Value represents the mean +/− standard deviations. ( B D ) Transepithelial electrical resistance measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set as 1.0. Values represent the mean of relative TEER +/− standard deviations. **** P

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection). (A C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A), 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days post-infection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque forming units (pfu) were plotted to the DPI. Value represents the mean +/− standard deviations. ( B D ) Transepithelial electrical resistance measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set as 1.0. Values represent the mean of relative TEER +/− standard deviations. **** P

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Incubation

    SARS-CoV-2 infection of primary human bronchial airway epithelium (HAE) is persistent. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque forming units (pfu) were determined (y-axis) and plotted to the dpi. Value represents the mean +/− standard deviations.

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: SARS-CoV-2 infection of primary human bronchial airway epithelium (HAE) is persistent. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque forming units (pfu) were determined (y-axis) and plotted to the dpi. Value represents the mean +/− standard deviations.

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection

    SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2 infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque forming units (pfu) were plotted to the dpi. Value represents the mean +/− standard deviations. (B) Transepithelial electrical resistance (TEER) measurement. The TEER of infected HAE-ALI B4-20 cultures were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set as 1.0. Values represent the mean of the relative TEER +/− standard deviations. n.s. indicates statistically no significance. (C D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( C ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( D ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue)

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2 infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque forming units (pfu) were plotted to the dpi. Value represents the mean +/− standard deviations. (B) Transepithelial electrical resistance (TEER) measurement. The TEER of infected HAE-ALI B4-20 cultures were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set as 1.0. Values represent the mean of the relative TEER +/− standard deviations. n.s. indicates statistically no significance. (C D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( C ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( D ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue)

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Immunofluorescence, Staining