sars cov np protein  (Sino Biological)


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    Name:
    SARS CoV Nucleoprotein NP Protein
    Description:
    A DNA sequence encoding the SARS CoV isolate Tor2 nucleoprotein NP 828858 1 Met1 Ala422 was expressed with a C terminal polyhistidine tag
    Catalog Number:
    40143-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus NP Protein SARS, coronavirus Nucleocapsid Protein SARS, coronavirus Nucleoprotein Protein SARS, cov np Protein SARS, ncov NP Protein SARS, novel coronavirus NP Protein SARS, novel coronavirus Nucleocapsid Protein SARS, novel coronavirus Nucleoprotein Protein SARS, NP Protein SARS, Nucleocapsid Protein SARS, Nucleoprotein Protein SARS
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological sars cov np protein
    Infectious virus shedding in ferrets. A/H1N1 virus (A), <t>SARS-CoV-2</t> (B) and SARS-CoV (C) titers were detected in throat (grey) and nasal (white) swabs collected from inoculated donor (bars) and indirect recipient (circles) ferrets. An individual donor-recipient pair is shown in each panel. Dotted line indicates detection limit.
    A DNA sequence encoding the SARS CoV isolate Tor2 nucleoprotein NP 828858 1 Met1 Ala422 was expressed with a C terminal polyhistidine tag
    https://www.bioz.com/result/sars cov np protein/product/Sino Biological
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov np protein - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance"

    Article Title: SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance

    Journal: bioRxiv

    doi: 10.1101/2020.10.19.345363

    Infectious virus shedding in ferrets. A/H1N1 virus (A), SARS-CoV-2 (B) and SARS-CoV (C) titers were detected in throat (grey) and nasal (white) swabs collected from inoculated donor (bars) and indirect recipient (circles) ferrets. An individual donor-recipient pair is shown in each panel. Dotted line indicates detection limit.
    Figure Legend Snippet: Infectious virus shedding in ferrets. A/H1N1 virus (A), SARS-CoV-2 (B) and SARS-CoV (C) titers were detected in throat (grey) and nasal (white) swabs collected from inoculated donor (bars) and indirect recipient (circles) ferrets. An individual donor-recipient pair is shown in each panel. Dotted line indicates detection limit.

    Techniques Used:

    Virus RNA shedding in ferrets. A/H1N1 (A), SARS-CoV-2 (B) and SARS-CoV (C) RNA was detected by qRT-PCR in throat (grey) and nasal (white) swabs collected from donor (bars) and recipient (circles) ferrets every other day. An individual donor-recipient pair is shown in each panel.
    Figure Legend Snippet: Virus RNA shedding in ferrets. A/H1N1 (A), SARS-CoV-2 (B) and SARS-CoV (C) RNA was detected by qRT-PCR in throat (grey) and nasal (white) swabs collected from donor (bars) and recipient (circles) ferrets every other day. An individual donor-recipient pair is shown in each panel.

    Techniques Used: Quantitative RT-PCR

    Detection of SARS-CoV RNA on the fur of donor ferrets. SARS-CoV RNA was detected by qRT-PCR in swabs collected from the fur on the left (dark grey) and right (light grey) flank of all four donor ferrets. Infectious virus was not detected in these samples.
    Figure Legend Snippet: Detection of SARS-CoV RNA on the fur of donor ferrets. SARS-CoV RNA was detected by qRT-PCR in swabs collected from the fur on the left (dark grey) and right (light grey) flank of all four donor ferrets. Infectious virus was not detected in these samples.

    Techniques Used: Quantitative RT-PCR

    Antibody responses in donor and recipient ferrets. Sera were collected from donor and recipient ferrets at the indicated days. Antibody responses against A/H1N1 virus (A) were measured by hemagglutination inhibition (HI) assay, whereas responses against SARS-CoV-2 (B) and SARS-CoV (C) were assessed using a nucleoprotein (NP) ELISA. Dotted lines indicate the detection limit of each assay.
    Figure Legend Snippet: Antibody responses in donor and recipient ferrets. Sera were collected from donor and recipient ferrets at the indicated days. Antibody responses against A/H1N1 virus (A) were measured by hemagglutination inhibition (HI) assay, whereas responses against SARS-CoV-2 (B) and SARS-CoV (C) were assessed using a nucleoprotein (NP) ELISA. Dotted lines indicate the detection limit of each assay.

    Techniques Used: HI Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins"

    Article Title: Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112868

    Sensitive and specific detection of the SARS-CoV-2 NP antigen. (a) Sensitivity of the sandwich LFIA for SARS-CoV-2 NP. Serially diluted SARS-CoV-2 NP (concentration range: 50 ng/reaction to 0.5 ng/reaction) was tested. After 20 min, the line intensities were analyzed using the portable LFIA reader ( I L : line intensity) and the LFIA strips were photographed using a smartphone. In addition, the intensity of the test and control lines was converted to a peak histogram by an image analyzer. (b) Intensity of the test lines measured by the portable LFIA reader. The limit of detection was calculated as the mean value of the negative controls plus three times the standard deviation. The 12H8(P C )/12H1(P D ) pair was the most sensitive for SARS-CoV-2 NP (as low as 2 ng of target antigen). (c) Sensitivity and selectivity of the best pair [12H8(P C )/12H1(P D )] for SARS-CoV-2 NP. Serially diluted cultured SARS-CoV-2 virus samples (concentration ranges: 2.5 × 10 5 pfu to 1 × 10 4 pfu) were used to evaluate the diagnostic performance of the LFIA biosensor. Moreover, NP from SARS-CoV, MERS-CoV, and influenza virus, or nasal swab specimens, were tested to investigate cross-reactivity (antigen concentration of all controls: 100 ng/reaction). After 20 min of sample flow, the intensities (I L ) of test lines were measured using the portable LFIA reader and each LFIA strip was photographed using a smartphone. (d) Ability of the LFIA biosensor to detect cultured SARS-CoV-2. Using the best pair [12H8(P C )/12H1(P D )], the LFIA biosensor successfully detected cultured SARS-CoV-2 levels as low as 1 × 10 4 pfu. (e) Cross-reactivity of the proposed LFIA biosensor with various negative controls. There was no cross-reactivity with SARS-CoV, MERS, influenza NP, or nasal swab specimens (antigen concentration of all controls: 100 ng/reaction).
    Figure Legend Snippet: Sensitive and specific detection of the SARS-CoV-2 NP antigen. (a) Sensitivity of the sandwich LFIA for SARS-CoV-2 NP. Serially diluted SARS-CoV-2 NP (concentration range: 50 ng/reaction to 0.5 ng/reaction) was tested. After 20 min, the line intensities were analyzed using the portable LFIA reader ( I L : line intensity) and the LFIA strips were photographed using a smartphone. In addition, the intensity of the test and control lines was converted to a peak histogram by an image analyzer. (b) Intensity of the test lines measured by the portable LFIA reader. The limit of detection was calculated as the mean value of the negative controls plus three times the standard deviation. The 12H8(P C )/12H1(P D ) pair was the most sensitive for SARS-CoV-2 NP (as low as 2 ng of target antigen). (c) Sensitivity and selectivity of the best pair [12H8(P C )/12H1(P D )] for SARS-CoV-2 NP. Serially diluted cultured SARS-CoV-2 virus samples (concentration ranges: 2.5 × 10 5 pfu to 1 × 10 4 pfu) were used to evaluate the diagnostic performance of the LFIA biosensor. Moreover, NP from SARS-CoV, MERS-CoV, and influenza virus, or nasal swab specimens, were tested to investigate cross-reactivity (antigen concentration of all controls: 100 ng/reaction). After 20 min of sample flow, the intensities (I L ) of test lines were measured using the portable LFIA reader and each LFIA strip was photographed using a smartphone. (d) Ability of the LFIA biosensor to detect cultured SARS-CoV-2. Using the best pair [12H8(P C )/12H1(P D )], the LFIA biosensor successfully detected cultured SARS-CoV-2 levels as low as 1 × 10 4 pfu. (e) Cross-reactivity of the proposed LFIA biosensor with various negative controls. There was no cross-reactivity with SARS-CoV, MERS, influenza NP, or nasal swab specimens (antigen concentration of all controls: 100 ng/reaction).

    Techniques Used: Concentration Assay, Standard Deviation, Cell Culture, Diagnostic Assay, Stripping Membranes

    A SARS-CoV-2-specific LFIA-based biosensor using scFv-Fc fusion proteins. (a) Schematic illustration of the development processes of SARS-CoV-2-specific scFv-Fc fusion proteins based on phage display technology. First, the SARS-CoV-2-specific scFv-Fc fusion proteins were screened, and four different scFv-Fcs with high affinity and specificity for the SARS-CoV-2 NP were selected using ELISAs. ( b) Schematic showing the scFv-Fc-based LFIA consisting of a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad. The selected scFv-Fc pairs were used as capture and detection antibodies. A test line placed on the NC membrane contains the capture scFv-Fc, and the CNB-conjugated detection scFv-Fc was immobilized on the conjugate pad. In the presence of SARS-CoV-2 NP, a sandwich complex (capture scFv-Fc-SARS-CoV-2 NP-detection scFv-Fc) was formed, and a clear red line appeared on the test line within 20 min. A handheld LFIA reader was used to analyze the line intensity semi-quantitatively. The proposed LFIA has is both sensitive and specific for SARS-CoV-2 NP, with no cross-reactivity with NPs from other coronaviruses such as MERS-CoV and SARS-CoV.
    Figure Legend Snippet: A SARS-CoV-2-specific LFIA-based biosensor using scFv-Fc fusion proteins. (a) Schematic illustration of the development processes of SARS-CoV-2-specific scFv-Fc fusion proteins based on phage display technology. First, the SARS-CoV-2-specific scFv-Fc fusion proteins were screened, and four different scFv-Fcs with high affinity and specificity for the SARS-CoV-2 NP were selected using ELISAs. ( b) Schematic showing the scFv-Fc-based LFIA consisting of a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad. The selected scFv-Fc pairs were used as capture and detection antibodies. A test line placed on the NC membrane contains the capture scFv-Fc, and the CNB-conjugated detection scFv-Fc was immobilized on the conjugate pad. In the presence of SARS-CoV-2 NP, a sandwich complex (capture scFv-Fc-SARS-CoV-2 NP-detection scFv-Fc) was formed, and a clear red line appeared on the test line within 20 min. A handheld LFIA reader was used to analyze the line intensity semi-quantitatively. The proposed LFIA has is both sensitive and specific for SARS-CoV-2 NP, with no cross-reactivity with NPs from other coronaviruses such as MERS-CoV and SARS-CoV.

    Techniques Used:

    Screening of SARS-CoV-2 NP-specific scFv-Fc fusion proteins. (a) Schematic illustration of the phage display screening process using a chicken naïve scFv antibody library. After three rounds of biopanning, positive clones were isolated by ELISA screening. (b) Selection of SARS-CoV-2 NP-specific binders. Of the 157 positive clones obtained from phage display screening, non-specific scFv binders showing a high background signal (n = 74), or cross-reactivity with MERS-CoV NP (n = 31) and SARS-CoV NP (n = 30), were eliminated; ultimately, 22 specific binders were isolated. (c) ELISA results for specific interactions between the scFv-Fc antibodies and the SARS-CoV-2 NP antigen. The purified scFv-Fc antibody bound specifically to SARS-CoV-2 NPs. (d) Western and dot blot assay results. 12H1, 12H8, and IG5 scFv-Fc antibodies bound strongly to SARS-CoV-2 NP. The scFv-Fc antibody bound weakly to SARS-CoV-2 NP in the dot blot assay.
    Figure Legend Snippet: Screening of SARS-CoV-2 NP-specific scFv-Fc fusion proteins. (a) Schematic illustration of the phage display screening process using a chicken naïve scFv antibody library. After three rounds of biopanning, positive clones were isolated by ELISA screening. (b) Selection of SARS-CoV-2 NP-specific binders. Of the 157 positive clones obtained from phage display screening, non-specific scFv binders showing a high background signal (n = 74), or cross-reactivity with MERS-CoV NP (n = 31) and SARS-CoV NP (n = 30), were eliminated; ultimately, 22 specific binders were isolated. (c) ELISA results for specific interactions between the scFv-Fc antibodies and the SARS-CoV-2 NP antigen. The purified scFv-Fc antibody bound specifically to SARS-CoV-2 NPs. (d) Western and dot blot assay results. 12H1, 12H8, and IG5 scFv-Fc antibodies bound strongly to SARS-CoV-2 NP. The scFv-Fc antibody bound weakly to SARS-CoV-2 NP in the dot blot assay.

    Techniques Used: Clone Assay, Isolation, Enzyme-linked Immunosorbent Assay, Selection, Purification, Western Blot, Dot Blot

    The biolayer interferometry (BLI) results of scFv-Fc antibodies against the SARS-CoV-2 NP antigen. (a) 12H1, (b) 12H8, (c) 12B3, and (d) 1G5. Dotted lines represent the response curves of BLI measurement, and solid lines represent the fitting curves based on the 1:1 binding model. Binding kinetics were measured for four different concentrates of each scFv-Fc antibody. K D : equilibrium dissociation constant; R 2 : coefficient of determination; k a : association rate constant; k d : dissociation rate constant.
    Figure Legend Snippet: The biolayer interferometry (BLI) results of scFv-Fc antibodies against the SARS-CoV-2 NP antigen. (a) 12H1, (b) 12H8, (c) 12B3, and (d) 1G5. Dotted lines represent the response curves of BLI measurement, and solid lines represent the fitting curves based on the 1:1 binding model. Binding kinetics were measured for four different concentrates of each scFv-Fc antibody. K D : equilibrium dissociation constant; R 2 : coefficient of determination; k a : association rate constant; k d : dissociation rate constant.

    Techniques Used: Binding Assay

    Identification of the sandwich pair that best detects the SARS-CoV-2 NP antigen. (a) Schematic diagram of the sandwich LFIA. A total of 12 pairs obtained from four different scFv-Fc antibodies were analyzed using a positive control (SARS-CoV-2 NP) and negative controls (SARS-CoV NP, MERS-CoV NP, and background). (b) Bar graph showing line intensity according to the target antigen (SARS-CoV-2 NP, SARS-CoV NP, MERS-CoV NP, and background) and sandwich pair (P C : capture probe; P D : detection probe). Among the 12 pairs, three optimal pairs [12H8(P C )/12H1(P D ); 12H8(P C )/12B3(P D ); and 12H8(P C )/1G5(P D )] were sensitive and specific for the SARS-CoV-2 NP. (c) Sandwich LFIA results for the three selected pairs. SARS-CoV-2 NP, SARS-CoV NP, MERS NP, and running buffer (negative control, NC) were introduced onto the LFIA strips. After 20 min of sample flow, the line intensities were measured using the portable LFIA reader ( I L : line intensity) and the LFIA strips were photographed using a smartphone.
    Figure Legend Snippet: Identification of the sandwich pair that best detects the SARS-CoV-2 NP antigen. (a) Schematic diagram of the sandwich LFIA. A total of 12 pairs obtained from four different scFv-Fc antibodies were analyzed using a positive control (SARS-CoV-2 NP) and negative controls (SARS-CoV NP, MERS-CoV NP, and background). (b) Bar graph showing line intensity according to the target antigen (SARS-CoV-2 NP, SARS-CoV NP, MERS-CoV NP, and background) and sandwich pair (P C : capture probe; P D : detection probe). Among the 12 pairs, three optimal pairs [12H8(P C )/12H1(P D ); 12H8(P C )/12B3(P D ); and 12H8(P C )/1G5(P D )] were sensitive and specific for the SARS-CoV-2 NP. (c) Sandwich LFIA results for the three selected pairs. SARS-CoV-2 NP, SARS-CoV NP, MERS NP, and running buffer (negative control, NC) were introduced onto the LFIA strips. After 20 min of sample flow, the line intensities were measured using the portable LFIA reader ( I L : line intensity) and the LFIA strips were photographed using a smartphone.

    Techniques Used: Positive Control, Negative Control

    Indirect LFIA results showing specific interactions between the scFv-Fc antibodies and SARS-CoV-2 NP. (a) Schematic diagram of the indirect LFIA using SARS-CoV-2 NP as a capture probe. Protein A-conjugated CNB c bound strongly to all antibodies in the sample buffer. The antibody-protein A-conjugated CNB complex binds to the pre-immobilized SARS-CoV-2 NP on the test line in a manner dependent upon the affinity between the antibody and SARS-CoV-2 NP. (b) Bar graph showing the intensities of the test lines. After 20 min of sample flow, the intensity of the test line was measured using a portable LFIA reader, and the line intensities were normalized using the following equation: Relative intensity = ( I L – I 0 )/ I 0 , I L : Line intensity in the presence of antibodies, I 0 : Line intensity in the absence of antibodies. (c) Results of the indirect LFIA. Three different NPs (derived from SARS-CoV-2, SARS-CoV, and MERS-CoV) were used for the test lines, and each scFv-Fc antibody was introduced onto the LFIA strip. After 20 min, the line intensities were measured using the portable LFIA reader ( I L : line intensity) and the LFIA strips were photographed with a smartphone.
    Figure Legend Snippet: Indirect LFIA results showing specific interactions between the scFv-Fc antibodies and SARS-CoV-2 NP. (a) Schematic diagram of the indirect LFIA using SARS-CoV-2 NP as a capture probe. Protein A-conjugated CNB c bound strongly to all antibodies in the sample buffer. The antibody-protein A-conjugated CNB complex binds to the pre-immobilized SARS-CoV-2 NP on the test line in a manner dependent upon the affinity between the antibody and SARS-CoV-2 NP. (b) Bar graph showing the intensities of the test lines. After 20 min of sample flow, the intensity of the test line was measured using a portable LFIA reader, and the line intensities were normalized using the following equation: Relative intensity = ( I L – I 0 )/ I 0 , I L : Line intensity in the presence of antibodies, I 0 : Line intensity in the absence of antibodies. (c) Results of the indirect LFIA. Three different NPs (derived from SARS-CoV-2, SARS-CoV, and MERS-CoV) were used for the test lines, and each scFv-Fc antibody was introduced onto the LFIA strip. After 20 min, the line intensities were measured using the portable LFIA reader ( I L : line intensity) and the LFIA strips were photographed with a smartphone.

    Techniques Used: Derivative Assay, Stripping Membranes

    3) Product Images from "Direct activation of endothelial cells by SARS-CoV-2 nucleocapsid protein is blocked by Simvastatin"

    Article Title: Direct activation of endothelial cells by SARS-CoV-2 nucleocapsid protein is blocked by Simvastatin

    Journal: bioRxiv

    doi: 10.1101/2021.02.14.431174

    The N protein from SARS-CoV2 but not the other coronaviruses potently induced endothelial cell activation. HLMECs were treated with or without five different recombinant viral N proteins (1 μg/mL) including SARS-CoV2, SARS-CoV, MERS-CoV, H7N9 and HKU1-CoV for 8 hrs. The expression of ICAM-1 and VCAM-1 was detected by western blot. TNFα (10 ng/mL) was served as a positive control. GAPDH was served as loading control. The experiments were repeated at least one more time.
    Figure Legend Snippet: The N protein from SARS-CoV2 but not the other coronaviruses potently induced endothelial cell activation. HLMECs were treated with or without five different recombinant viral N proteins (1 μg/mL) including SARS-CoV2, SARS-CoV, MERS-CoV, H7N9 and HKU1-CoV for 8 hrs. The expression of ICAM-1 and VCAM-1 was detected by western blot. TNFα (10 ng/mL) was served as a positive control. GAPDH was served as loading control. The experiments were repeated at least one more time.

    Techniques Used: Activation Assay, Recombinant, Expressing, Western Blot, Positive Control

    4) Product Images from "Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients"

    Article Title: Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2607.200841

    Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

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

    Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT 50 , plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT 50 , plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Neutralization, Plaque Reduction Neutralization Test, Binding Assay, Fluorescence

    5) Product Images from "Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients"

    Article Title: Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2607.200841

    Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

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

    Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT 50 , plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT 50 , plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Neutralization, Plaque Reduction Neutralization Test, Binding Assay, Fluorescence

    6) Product Images from "Potent human neutralizing antibodies elicited by SARS-CoV-2 infection"

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.03.21.990770

    Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green
    Figure Legend Snippet: Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green

    Techniques Used: Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Sequencing

    Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.
    Figure Legend Snippet: Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.

    Techniques Used: Binding Assay, SPR Assay, Incubation

    Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.
    Figure Legend Snippet: Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Staining, Labeling, FACS, Positive Control, Negative Control

    Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.
    Figure Legend Snippet: Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.

    Techniques Used: Neutralization, Enzyme-linked Immunosorbent Assay, Serial Dilution, Binding Assay, Activity Assay

    Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A). All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
    Figure Legend Snippet: Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A). All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Derivative Assay, Isolation, Sequencing

    ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.
    Figure Legend Snippet: ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Sampling, Sequencing, Binding Assay, Activity Assay

    Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.
    Figure Legend Snippet: Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.

    Techniques Used: Binding Assay, Isolation, SPR Assay, Purification, Chromatin Immunoprecipitation, Injection, Derivative Assay

    7) Product Images from "A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering"

    Article Title: A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427310

    Immune responses of T4-SARS-CoV-2 immunized mice. a. Anti-RBD IgG antibody titers in the sera from group G5 (T4-HocΔ-SocΔ-S-ecto-Ee-NP) at weeks 2 (prime), 5 (boost-1), and 8 (boost-2). For boost-2, T4-S-trimers particles were used. **P
    Figure Legend Snippet: Immune responses of T4-SARS-CoV-2 immunized mice. a. Anti-RBD IgG antibody titers in the sera from group G5 (T4-HocΔ-SocΔ-S-ecto-Ee-NP) at weeks 2 (prime), 5 (boost-1), and 8 (boost-2). For boost-2, T4-S-trimers particles were used. **P

    Techniques Used: Mouse Assay

    Immunogenicity and protective efficacy of T4-SARS-CoV-2 vaccine candidates in mice. a. Schematic diagram showing BALB/c mice immunized by the intramuscular (i.m.) route using T4-SARS-CoV-2 vaccine formulations. b. I. Formulations and mouse groups used for vaccinations. HSΔ indicates Hoc deletion and Soc deletion. Blue color (S-ecto, S-fl, and RBD) indicates the insertion of mammalian gene expression cassette into T4 genome as DNA vaccine. Red color indicates the capsid-displayed Ee, S-trimers, or E.coli -produced rRBD or sRBD protein, or the capsid-encapsidated NP protein. Naïve mice and mice immunized with the phage lacking any CoV-2 genes served as negative controls whereas mice immunized with S-trimers adjuvanted with Alhydrogel served as a positive control. II. Prime-boost immunization scheme. BALB/c mice (5 per group) were immunized on weeks 0, 3, and 6 and challenged intranasally (i.n.) with a mouse-adapted SARS-CoV-2 strain (SARS-CoV-2 MA10) 47 on week 14. c to f . The boost-2 sera (week 8 bleeding) from various groups were assessed by ELISA for antigen-specific IgG antibody titers (endpoint) against S-ecto (c), RBD (d), NP (e), and E (f). *P
    Figure Legend Snippet: Immunogenicity and protective efficacy of T4-SARS-CoV-2 vaccine candidates in mice. a. Schematic diagram showing BALB/c mice immunized by the intramuscular (i.m.) route using T4-SARS-CoV-2 vaccine formulations. b. I. Formulations and mouse groups used for vaccinations. HSΔ indicates Hoc deletion and Soc deletion. Blue color (S-ecto, S-fl, and RBD) indicates the insertion of mammalian gene expression cassette into T4 genome as DNA vaccine. Red color indicates the capsid-displayed Ee, S-trimers, or E.coli -produced rRBD or sRBD protein, or the capsid-encapsidated NP protein. Naïve mice and mice immunized with the phage lacking any CoV-2 genes served as negative controls whereas mice immunized with S-trimers adjuvanted with Alhydrogel served as a positive control. II. Prime-boost immunization scheme. BALB/c mice (5 per group) were immunized on weeks 0, 3, and 6 and challenged intranasally (i.n.) with a mouse-adapted SARS-CoV-2 strain (SARS-CoV-2 MA10) 47 on week 14. c to f . The boost-2 sera (week 8 bleeding) from various groups were assessed by ELISA for antigen-specific IgG antibody titers (endpoint) against S-ecto (c), RBD (d), NP (e), and E (f). *P

    Techniques Used: Mouse Assay, Expressing, Produced, Positive Control, Enzyme-linked Immunosorbent Assay

    A pipeline of SARS-CoV-2 vaccine candidates generated by sequential CRISPR engineering. a. Schematic showing a representative sequence in which the WT phage was used as a starting infection of CRISPR E. coli containing spacer 1 and donor 1. The resultant T4-mutant 1 (T4-M1) was used to infect bacteria containing spacer 2 and donor 2 to produce recombinant T4-mutant 2 (T4-M2) which has two insertion/deletion mutations, and so forth. By sequential CRISPR engineering and simple phage infections, recombinant phages with multiple desired mutations were created. Each color on phage capsid here represents a mutation. b. One example of sequential phage CRISPR engineering for creating the T4-SARS-CoV-2 nanovaccine. Numerous CoV-2 components, including CAGpromoter-S-ecto insertion, CAGpromoter-S-fl insertion, CMVpromoter-RBD insertion, Hoc deletion, Ee-Hoc insertion, Ec-Hoc insertion, Soc deletion, Soc-sRBD display, M21-Soc-sRBD display, Soc-SpyCatcher display, refolding SUMO-RBD-Spy display, S-trimer display, IPIII deletion, IPII deletion, and NP encapsidation, were permutated and combined as needed. The resultant SARS-CoV-2 vaccine candidates were characterized by PCR, DNA sequencing and/or SDS-PAGE, and some of these were then tested in a mouse study. M21 indicates a potential T cell 21 aa epitope (SYFIASFRLFARTRSMWSFNP) from SARS-CoV-2 membrane protein. c. WB showing NP protein encapsidation in the phages containing CTSam-NP insertion at IPIII deletion site.
    Figure Legend Snippet: A pipeline of SARS-CoV-2 vaccine candidates generated by sequential CRISPR engineering. a. Schematic showing a representative sequence in which the WT phage was used as a starting infection of CRISPR E. coli containing spacer 1 and donor 1. The resultant T4-mutant 1 (T4-M1) was used to infect bacteria containing spacer 2 and donor 2 to produce recombinant T4-mutant 2 (T4-M2) which has two insertion/deletion mutations, and so forth. By sequential CRISPR engineering and simple phage infections, recombinant phages with multiple desired mutations were created. Each color on phage capsid here represents a mutation. b. One example of sequential phage CRISPR engineering for creating the T4-SARS-CoV-2 nanovaccine. Numerous CoV-2 components, including CAGpromoter-S-ecto insertion, CAGpromoter-S-fl insertion, CMVpromoter-RBD insertion, Hoc deletion, Ee-Hoc insertion, Ec-Hoc insertion, Soc deletion, Soc-sRBD display, M21-Soc-sRBD display, Soc-SpyCatcher display, refolding SUMO-RBD-Spy display, S-trimer display, IPIII deletion, IPII deletion, and NP encapsidation, were permutated and combined as needed. The resultant SARS-CoV-2 vaccine candidates were characterized by PCR, DNA sequencing and/or SDS-PAGE, and some of these were then tested in a mouse study. M21 indicates a potential T cell 21 aa epitope (SYFIASFRLFARTRSMWSFNP) from SARS-CoV-2 membrane protein. c. WB showing NP protein encapsidation in the phages containing CTSam-NP insertion at IPIII deletion site.

    Techniques Used: Generated, CRISPR, Sequencing, Infection, Mutagenesis, Recombinant, Polymerase Chain Reaction, DNA Sequencing, SDS Page, Western Blot

    Serum antibody responses in various T4-SARS-CoV-2 immunized mice. a and b. Anti-S-ecto IgG1 (a) and IgG2a (b) antibody titers in the boost-2 sera (week 8 bleeding) from various groups. c and d. Anti-RBD IgG1 (c) and IgG2a (d) antibody titers in the boost-2 sera. e and f . Anti-NP IgG1 (e) and IgG2a (f) antibody titers in the boost-2 sera. g and h . Anti-E IgG1 (g) and IgG2a (h) antibody titers in the boost-2 sera. *P
    Figure Legend Snippet: Serum antibody responses in various T4-SARS-CoV-2 immunized mice. a and b. Anti-S-ecto IgG1 (a) and IgG2a (b) antibody titers in the boost-2 sera (week 8 bleeding) from various groups. c and d. Anti-RBD IgG1 (c) and IgG2a (d) antibody titers in the boost-2 sera. e and f . Anti-NP IgG1 (e) and IgG2a (f) antibody titers in the boost-2 sera. g and h . Anti-E IgG1 (g) and IgG2a (h) antibody titers in the boost-2 sera. *P

    Techniques Used: Mouse Assay

    Virus neutralization titers of rabbit sera. Infection of Vero E6 cells by live SARS-CoV-2 US-WA-1/2020 was determined in the presence of rabbit sera at a series of two-fold dilutions starting from 1:4. Culture medium only and CoV-2 virus only were used as negative and positive controls, respectively. R1442 to R1457 refer to tag numbers of rabbits. The data in control groups were presented as means ± SD of 32 wells. The data in rabbit sera groups were shown as means of duplicates.
    Figure Legend Snippet: Virus neutralization titers of rabbit sera. Infection of Vero E6 cells by live SARS-CoV-2 US-WA-1/2020 was determined in the presence of rabbit sera at a series of two-fold dilutions starting from 1:4. Culture medium only and CoV-2 virus only were used as negative and positive controls, respectively. R1442 to R1457 refer to tag numbers of rabbits. The data in control groups were presented as means ± SD of 32 wells. The data in rabbit sera groups were shown as means of duplicates.

    Techniques Used: Neutralization, Infection

    Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.
    Figure Legend Snippet: Construction and screening of various truncated SARS-CoV-2 RBDs. a. Structural models of recombinant WT RBD and various truncated RBDs bound to human ACE2. ACE2 is shown in green. The truncated RBD clones are shown in red and the WT RBD and deleted regions are shown in cyan. The Protein Data Bank (PDB) code for the SARS-CoV-2 RBD–ACE2 complex is 6M0J 34 . The truncated RBDs were generated using Chimera software. b. Solubility analysis of Soc-fused truncated RBDs after cloning and expression in E. coli under the control of the phage T7 promoter. After lysis of E. coli and centrifugation, the supernatant and pellet were analyzed by SDS-PAGE. The presence of Soc-truncated RBDs in the pellet and their absence in the supernatant demonstrated insolubility. The red arrowheads indicate the band positions of various Soc-truncated RBDs.

    Techniques Used: Recombinant, Clone Assay, Generated, Software, Solubility, Expressing, Lysis, Centrifugation, SDS Page

    Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P
    Figure Legend Snippet: Incorporation of various SARS-CoV-2 vaccine payloads into phage T4 nanoparticle. a. Schematic showing steps in T4 phage head morphogenesis. Mem, E. coli membrane; CTS, capsid targeting sequence. b and c. SDS-PAGE and Western Blot (WB) analysis of phage particles with IPII and IPIII deletions (IPIIΔIPIIIΔ) and NP encapsidation. Since NP has a very similar molecular size to T4 major capsid protein gp23*, an NP-specific antibody was used to detect NP. d. Structural model of viroporin-like tetrameric assembly of CoV-2 E protein 32 . The N-terminal seven residues and C-terminal ten residues are not shown due to the lack of a corresponding segment in the structural template used for homology modeling. Ee* indicates amino acids (aa) 8-12 and Ec* indicates aa 53-65. e. SDS-PAGE of Hoc deletion and Soc deletion phage (HocΔSocΔ). f. SDS-PAGE of recombinant phages displaying Ee-Hoc or Ec-Hoc fusion proteins. g. Schematic showing Soc-sRBD or Soc-SpyCatcher (SpyC) in vivo display on T4-SocΔ capsid. Soc-sRBD or Soc-SpyCatcher expression under the control of phage T7 promoter was induced by IPTG. Most of the expressed Soc-RBD was in the inclusion body (IB). Soluble Soc-sRBD (minor amount) or Soc-SpyC can be efficiently displayed on capsid. h. SDS-PAGE showing ~100 copies of Soc-sRBD displayed on T4 capsid. i. SDS-PAGE showing ~500 copies of Soc-SpyCatcher displayed on T4 capsid. j. Schematic diagram showing the solubilization and refolding of SUMO (small ubiquitin like modifiers)-RBD-Spytag inclusion body. Refolded SUMO-RBD-Spytag (rRBD) protein was efficiently displayed on T4-SpyCatcher phage via Spytag-SpyCatcher bridging. k. Display of rRBD on the T4-SpyCacher surface at increasing ratios of rRBD molecules to capsid Soc binding sites (0:1 to 2:1). RBD specific antibody was used to verify the displayed rRBD and rRBD-SpyCatcher-Soc complexes. T4* indicates T4-S-ecto-NP-Ec-SocΔ recombinant phage. Blue and red arrows indicate rRBD/complexes and Soc-SpyCatcher, respectively. l to o . Comparison of binding of T4-sRBD, and T4-rRBD phages to soluble human ACE2 receptor (l), monoclonal antibody (mAb) 1 (human IgG Clone #bcb03, Thermo Fisher) (m), mAb2 (rabbit IgG Clone #007, Sino Bio) (n), and polyclonal antibodies (pAb) (rabbit PAb, Sino Bio) (o) using BSA and T4 phage as controls. p. Comparison of binding of E. coli -produced rRBD to human ACE2 with the HEK293-produced RBD. **P

    Techniques Used: Sequencing, SDS Page, Western Blot, Recombinant, In Vivo, Expressing, Binding Assay, Produced

    Design of T4-SARS-CoV-2 nanovaccine by CRISPR engineering. Engineered DNAs corresponding to various components of SARS-CoV-2 virion are incorporated into bacteriophage T4 genome. Each DNA was introduced into E. coli as a donor plasmid (a) , recombined into injected phage genome through CRISPR-targeted genome editing (b) . Different combinations of CoV-2 inserts were then generated by simple phage infections and identifying the recombinant phages in the progeny (c) . For example, recombinant phage containing CoV-2 insert #1 (dark blue) can be used to infect CRISPR E. coli containing Co-V2 insert containing donor plasmid #2 (dark red). The progeny plaques obtained will contain recombinant phage #3 with both inserts #1 and #2 (dark blue plus dark red) in the same genome. This process was repeated to rapidly construct a pipeline of multiplex T4-SARS-CoV-2 vaccine phages (d) . Selected vaccine candidates were then screened in a mouse model (e) to identify the most potent vaccine (f) . Structural model of T4-SARS-CoV-2 Nanovaccine showing an enlarged view of a single hexameric capsomer (g) . The capsomer shows six subunits of major capsid protein gp23* (green), trimers of Soc (blue), and a Hoc fiber (yellow) at the center of capsomer. The expressible spike genes are inserted into phage genome, the 12 aa E external peptide (red) is displayed at the tip of Hoc fiber, S-trimers (cyan) are attached to Soc subunits, and nucleocapsid proteins (yellow) are packaged in genome core. See Results , Materials and Methods, and Supplementary Video for additional details.
    Figure Legend Snippet: Design of T4-SARS-CoV-2 nanovaccine by CRISPR engineering. Engineered DNAs corresponding to various components of SARS-CoV-2 virion are incorporated into bacteriophage T4 genome. Each DNA was introduced into E. coli as a donor plasmid (a) , recombined into injected phage genome through CRISPR-targeted genome editing (b) . Different combinations of CoV-2 inserts were then generated by simple phage infections and identifying the recombinant phages in the progeny (c) . For example, recombinant phage containing CoV-2 insert #1 (dark blue) can be used to infect CRISPR E. coli containing Co-V2 insert containing donor plasmid #2 (dark red). The progeny plaques obtained will contain recombinant phage #3 with both inserts #1 and #2 (dark blue plus dark red) in the same genome. This process was repeated to rapidly construct a pipeline of multiplex T4-SARS-CoV-2 vaccine phages (d) . Selected vaccine candidates were then screened in a mouse model (e) to identify the most potent vaccine (f) . Structural model of T4-SARS-CoV-2 Nanovaccine showing an enlarged view of a single hexameric capsomer (g) . The capsomer shows six subunits of major capsid protein gp23* (green), trimers of Soc (blue), and a Hoc fiber (yellow) at the center of capsomer. The expressible spike genes are inserted into phage genome, the 12 aa E external peptide (red) is displayed at the tip of Hoc fiber, S-trimers (cyan) are attached to Soc subunits, and nucleocapsid proteins (yellow) are packaged in genome core. See Results , Materials and Methods, and Supplementary Video for additional details.

    Techniques Used: CRISPR, Plasmid Preparation, Injection, Generated, Recombinant, Construct, Multiplex Assay

    Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering. a. Schematic of T4 CRISPR engineering. b. Four nonessential regions of T4 genome are chosen for deletion and insertion of various SARS-CoV-2 genes (shown in red; SegF/Soc, FarP, IP, and Hoc). 6P, six proline substitutions in S-ecto (F817P, A892P, A899P, A942P, K986P, and V987P). Fol, T4 fibritin motif Foldon for efficient trimerization. Tag, octa-histidine and twin-strep tags. Furin cleavage site RRAR was mutated to GSAS to stabilize trimers in a prefusion state 31 . c. Efficiency of plating (EOP) of representative Cpf1-FarP7K and Cpf1-SegF spacers. d. Plate showing plaques from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer plus S-ecto donor. e. Recombination frequency of three spike gene (RBD, S-ecto, and S-fl) insertions. f. DNA sequencing of thirty independent plaques showed that > 95% of the plaques generated in S-ecto recombination contained the correct S-ecto insert. g. Plate showing that the wild-type (WT), T4-RBD, T4-S-fl, T4-S-ecto, and T4-(S-ecto)-RBD recombinant phages had similar plaque size.
    Figure Legend Snippet: Construction of T4-SARS-CoV-2 recombinant phages by CRISPR engineering. a. Schematic of T4 CRISPR engineering. b. Four nonessential regions of T4 genome are chosen for deletion and insertion of various SARS-CoV-2 genes (shown in red; SegF/Soc, FarP, IP, and Hoc). 6P, six proline substitutions in S-ecto (F817P, A892P, A899P, A942P, K986P, and V987P). Fol, T4 fibritin motif Foldon for efficient trimerization. Tag, octa-histidine and twin-strep tags. Furin cleavage site RRAR was mutated to GSAS to stabilize trimers in a prefusion state 31 . c. Efficiency of plating (EOP) of representative Cpf1-FarP7K and Cpf1-SegF spacers. d. Plate showing plaques from phage infection of bacteria containing Cpf1-FarP7K spacer only, S-ecto donor only, or Cpf1-FarP7K spacer plus S-ecto donor. e. Recombination frequency of three spike gene (RBD, S-ecto, and S-fl) insertions. f. DNA sequencing of thirty independent plaques showed that > 95% of the plaques generated in S-ecto recombination contained the correct S-ecto insert. g. Plate showing that the wild-type (WT), T4-RBD, T4-S-fl, T4-S-ecto, and T4-(S-ecto)-RBD recombinant phages had similar plaque size.

    Techniques Used: Recombinant, CRISPR, Infection, DNA Sequencing, Generated

    CRISPR engineering of non-essential T4 genome. a. Schematic showing the 18-kb nonessential segment FarP and 11-kb nonessential segment 39-56 on T4 genome. b. Plaque size of wild-type (WT), T4- FarP 18 kb del. , T4- 39-56 11 kb del. , and T4- FarP 39-56 29 kb del. phages. Note the small size of T4- 39-56 11 kb del. and T4- FarP 39-56 29 kb del. plaques. c. Structural models of SARS-CoV-2 virus, spike trimer, and receptor binding domain (RBD). d. Schematics of S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome. e. Efficiency of plating of three sets of Cpf1-FarP7K spacers and three sets of Cpf1-SegF spacers. f. Efficiency of plating of various spacers used for T4 genome engineering in this study.
    Figure Legend Snippet: CRISPR engineering of non-essential T4 genome. a. Schematic showing the 18-kb nonessential segment FarP and 11-kb nonessential segment 39-56 on T4 genome. b. Plaque size of wild-type (WT), T4- FarP 18 kb del. , T4- 39-56 11 kb del. , and T4- FarP 39-56 29 kb del. phages. Note the small size of T4- 39-56 11 kb del. and T4- FarP 39-56 29 kb del. plaques. c. Structural models of SARS-CoV-2 virus, spike trimer, and receptor binding domain (RBD). d. Schematics of S-full length (S-fl) and S-ectodomain (S-ecto) expression cassettes used for insertion into T4 genome. e. Efficiency of plating of three sets of Cpf1-FarP7K spacers and three sets of Cpf1-SegF spacers. f. Efficiency of plating of various spacers used for T4 genome engineering in this study.

    Techniques Used: CRISPR, Binding Assay, Expressing

    8) Product Images from "Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2"

    Article Title: Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2

    Journal: Viruses

    doi: 10.3390/v12070699

    Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.
    Figure Legend Snippet: Analysis of the effect of Fr 1C on the SARS-CoV-2 proteins and genome. DMSO and Fr 1C were added to cell culture supernatants containing SARS-CoV-2 and were incubated at 25 °C for 48 h. n = 3 per group. ( A,B ) The images are the results of WB to detect SARS-CoV-2 ( A ) S2 subunit protein and ( B ) NP. ( C ) The image is the result of RT–PCR using NIID_2019-nCoV_N_F2 and R2 primers which amplify 158 bp region on SARS-CoV-2 gene. M: Marker.

    Techniques Used: Cell Culture, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Marker

    9) Product Images from "Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients"

    Article Title: Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2607.200841

    Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Validation of use of S1 (A, B), RBD (C), and N protein (D) ELISAs for detection of SARS-CoV-2–specific antibodies infections. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

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

    Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT 50 , plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.
    Figure Legend Snippet: Validation of 2 commercial ELISAs for detection of SARS-CoV-2–specific IgG (A, C, E, G) and IgA (B, D, F, H). A, B) Validation of the specificity of the 2 ELISA platforms; C, D) kinetics of antibody responses in 3 COVID-19 patients; E, F) cross-reactivity of HCoV-OC43 serum samples in commercial platforms; G, H) correlation between antibody responses detected by the ELISAs and the plaque reduction neutralization assay. Gray dots indicate specificity cohorts A–C, including healthy blood donors (n = 45), non-CoV respiratory infections (n = 76), and HCoV infections (n = 75); blue dots indicate non-SARS-CoV-2 zoonotic coronavirus infections (i.e., MERS-CoV [n = 7] and SARS-CoV [n = 2]); red dots indicate patients with severe COVID-19; and green and black dots indicate patients with mild COVID-19. Dotted horizontal lines indicate ELISA cutoff values. CoV, coronavirus; COVID-19, coronavirus disease 2019; HCoV, human coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; N, nucleocapsid; OD, optical density; PRNT 50 , plaque reduction neutralization assay; RBD, receptor-binding domain; RFU, relative fluorescence unit; S, spike; SARS-CoV-2; severe acute respiratory syndrome coronavirus 2.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Neutralization, Plaque Reduction Neutralization Test, Binding Assay, Fluorescence

    10) Product Images from "Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2"

    Article Title: Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.604663

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the pre-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.
    Figure Legend Snippet: Binding activity of the plant-produced ACE2-Fc with the commercial receptor binding domain of SARS-CoV-2 (SARS-CoV-2 RBD) from Sf9 cells was analyzed by ELISA. PBS buffer and S1 protein of PEDV were used as negative controls. Data are presented as mean ± SD of triplicates.

    Techniques Used: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay

    Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.
    Figure Legend Snippet: Dose-dependent effect of plant-produced ACE2-Fc on SARS-CoV-2 inhibition and neutralization at the post-infection phase. Experimental design of plant-produced ACE2-Fc and SARS-CoV-2 mixture added to Vero E6 cells (at 25TCID 50 ) (A) . SARS-CoV-2 infection profiles in Vero E6 cells which were treated with eight concentrations of plant-produced ACE2-Fc (B) . Percentage of SARS-CoV-2 inhibition in Vero E6 cells, which were treated with eight concentrations of plant-produced ACE2-Fc starting with 200 μg/ml (C) . Efficacy of SARS-CoV-2 inhibition in Vero E6 cells, which were treated by eight concentrations of plant-produced ACE2-Fc (D) . The data were showed as mean ± SD of triplicates in individual concentrations.

    Techniques Used: Produced, Inhibition, Neutralization, Infection

    Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .
    Figure Legend Snippet: Schematic representation of plant expression vector pBYR2e-ACE2-Fc used in the present study (A) . Diagrammatic representation showing the binding of plant-produced ACE2-Fc with SARS-CoV-2 thereby preventing the virus entry into the host cell (B) .

    Techniques Used: Expressing, Plasmid Preparation, Binding Assay, Produced

    11) Product Images from "COVID-19 in Autoinflammatory Diseases with Immunosuppressive Treatment"

    Article Title: COVID-19 in Autoinflammatory Diseases with Immunosuppressive Treatment

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm10040605

    Schematic overview of the pathogenesis for IL-1-mediated autoimmune disease (AID) and COVID-19. (1.) Pathogenesis of NLRP3 Inflammasome associated AID ( gray ): Inflammasome formation is induced by a variety of triggers. Activated NLRP3 subsequently drives caspase-1 activation. Caspase-1 mediates transformation from pro-IL-1β and pro-IL-18 to active IL-1β and IL-18. The positive feedback loop stimulates NF-kB. (2.) SARS-CoV-2 pathogenesis ( white ): SARS-CoV-2 can stimulate a hyperinflammatory immune response with epithelial cell-mediated production of reactive oxygen species (ROS). ROS can stimulate NF-kB and NLRP3. Both pathways (1. and 2.) result in increased cytokine levels with laboratory signs and clinical symptoms associated with hypercytokinemia. Abbreviations: SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; COVID-19: coronavirus disease 2019; ROS: reactive oxygen species; NLRP3: (NOD)-like receptor protein 3; NF-kB: nuclear factor kappa B; IL: interleukin; CRP: C-reactive protein, ESR: Erythrocyte sedimentation rate, MAS: macrophage activation syndrome; CSS: cytokine storm syndrome.
    Figure Legend Snippet: Schematic overview of the pathogenesis for IL-1-mediated autoimmune disease (AID) and COVID-19. (1.) Pathogenesis of NLRP3 Inflammasome associated AID ( gray ): Inflammasome formation is induced by a variety of triggers. Activated NLRP3 subsequently drives caspase-1 activation. Caspase-1 mediates transformation from pro-IL-1β and pro-IL-18 to active IL-1β and IL-18. The positive feedback loop stimulates NF-kB. (2.) SARS-CoV-2 pathogenesis ( white ): SARS-CoV-2 can stimulate a hyperinflammatory immune response with epithelial cell-mediated production of reactive oxygen species (ROS). ROS can stimulate NF-kB and NLRP3. Both pathways (1. and 2.) result in increased cytokine levels with laboratory signs and clinical symptoms associated with hypercytokinemia. Abbreviations: SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; COVID-19: coronavirus disease 2019; ROS: reactive oxygen species; NLRP3: (NOD)-like receptor protein 3; NF-kB: nuclear factor kappa B; IL: interleukin; CRP: C-reactive protein, ESR: Erythrocyte sedimentation rate, MAS: macrophage activation syndrome; CSS: cytokine storm syndrome.

    Techniques Used: Activation Assay, Transformation Assay, Sedimentation

    12) Product Images from "Potent human neutralizing antibodies elicited by SARS-CoV-2 infection"

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2020.03.21.990770

    Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green
    Figure Legend Snippet: Heavy chain repertoires of SARS-CoV-2 RBD-specific antibodies analyzed (A) by individual subject or (B) across the eight subjects. (A) Distribution and frequency of heavy chain variable (VH) genes usage in each subject shown along the horizontal bar. The same color scheme is used for each VH family across all study subjects. The VHs that dominate across isolated antibodies are indicated by actual frequencies in their respective color boxes. The number of RBD-binding antibodies versus total antibodies isolated are shown on the right. (B) Clustering of VH genes and their association with ELISA binding activity across the eight subjects. Unrooted phylogenetic tree depicting the genetic relationships among all VH genes of the RBD-binding antibodies. Branch lengths are drawn to scale so that sequence relatedness can be readily assessed. Sequences from the same study subject are shown in the same color at the branch tips. Colored circles represent the proportion (light orange, > 80%; light yellow, 60%-80%; light green

    Techniques Used: Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Sequencing

    Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.
    Figure Legend Snippet: Antibody and ACE2 competition for binding to SARS-CoV-2 RBD measured by SPR. The sensorgrams show distinct binding patterns of ACE2 to SARS-CoV-2 RBD with (red curve) or without (black curve) prior incubation with each testing antibody. The competition capacity of each antibody is indicated by the level of reduction in response unit of ACE2 comparing with or without prior antibody incubation.

    Techniques Used: Binding Assay, SPR Assay, Incubation

    Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.
    Figure Legend Snippet: Analysis of plasma binding to cell surface expressed trimeric Spike protein. HEK 293T cells transfected with expression plasmid encoding the full length spike of SARS-CoV-2, SARS-CoV or MERS-CoV were incubated with 1:100 dilutions of plasma from the study subjects. The cells were then stained with PE labeled anti-human IgG Fc secondary antibody and analyzed by FACS. Positive control antibodies include S230 and m396 targeting the RBD of SARS-CoV Spike, and Mab-GD33 targeting the RBD of MERS-CoV Spike. VRC01 is negative control antibody targeting HIV-1 envelope glycoprotein.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Staining, Labeling, FACS, Positive Control, Negative Control

    Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.
    Figure Legend Snippet: Antibody neutralization analyzed by pseudovirus and live SARS-CoV-2. (A) Quality control of antibody through ELISA analysis prior to neutralization assay. A serial dilution of each antibody was evaluated against SARS-CoV-2 RBD coated on the ELISA plate and their binding activity was recorded at an optical density (OD) of 450nm and 630nm. (B-C) Antibody neutralization analyzed by pseudovirus (B) or live SARS-CoV-2 (C). A serial dilution of each antibody was tested against pseudovirus while two dilutions against live SARS-CoV-2. Cytopathic effects (CPE) were observed daily and recorded on Day 2 post-exposure. Selected antibodies and their concentrations tested are indicated at the upper left corner.

    Techniques Used: Neutralization, Enzyme-linked Immunosorbent Assay, Serial Dilution, Binding Assay, Activity Assay

    Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A). All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.
    Figure Legend Snippet: Analyses of plasma and B cell responses specific to SARS-CoV-2. Serial dilutions of plasma samples were analyzed for binding to the (A) RBDs or (B) trimeric Spikes of SARS-CoV-2, SARS-CoV and MERS-CoV by ELISA and (C) for neutralizing activity against pseudoviruses bearing envelope glycoprotein of SARS-CoV-2, SARS-CoV and MERS-CoV. Binding to SARS-CoV-2 NP protein was also evaluated (A). All results were derived from at least two independent experiments. (D) Gating strategy for analysis and isolation of RBD-specific memory B cells and (E) their representation among the total and memory subpopulation of B cells in the eight study subjects. Samples were named as either A, B, or C depending on collection sequence. FSC-W, forward scatter width; FSC-A, forward scatter area; and SSC-A side scatter area.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Derivative Assay, Isolation, Sequencing

    ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.
    Figure Legend Snippet: ELISA screening of SARS-CoV-2 RBD-specific antibodies in the supernatant of transfected cells. The study subjects and the date of sampling are indicated on the top. Samples were named as either A, B, or C depending on collection sequence. Antibodies tested for each sample are aligned in one vertical column whenever possible. For each evaluated antibody, at least two independent measurements were performed and are presented adjacently on the same row. Binding activities were assessed by OD 450 and indicated by the color scheme on the right. Negatives (no binding activity) are shown in gray for OD 450 values less than 0.1.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Sampling, Sequencing, Binding Assay, Activity Assay

    Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.
    Figure Legend Snippet: Binding kinetics of isolated mAbs with SARS-CoV-2 RBD measured by SPR. The purified soluble SARS-CoV-2 RBD were covalently immobilized onto a CM5 sensor chip followed by injection of individual antibody at four or five different concentrations. The black lines indicate the experimentally derived curves while the red lines represent fitted curves based on the experimental data.

    Techniques Used: Binding Assay, Isolation, SPR Assay, Purification, Chromatin Immunoprecipitation, Injection, Derivative Assay

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    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
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    Clone Assay:

    Article Title: Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins
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    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
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    Recombinant:

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
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    Derivative Assay:

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    Incubation:

    Article Title: Potent human neutralizing antibodies elicited by SARS-CoV-2 infection
    Article Snippet: Secreted RBD and trimeric Spike were harvested from the supernatant and purified by gel filtration chromatography as previously reported , , - . .. ELISA analysis of plasma and antibody binding to RBD, trimeric Spike, and NP proteinsThe recombinant RBDs and trimeric Spike derived from SARS-CoV-2, SARS-CoV and MERS-CoV and the SARS-CoV-2 NP protein (Sino Biological, Beijing) were diluted to final concentrations of 0.5 μg/ml or 2μg/ml, then coated onto 96-well plates and incubated at 4°C overnight. .. Samples were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at RT for 1h.

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    Sino Biological sars cov 2
    Affinity screening of the calibration antibodies. (A) Calibration curves of 4 different monoclonal humanized S1 specific IgG against the S1 protein from <t>SARS-CoV-2.</t> (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.
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    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: They were also believed to have cross-reactivities with the S1 protein of SARS-CoV-2.

    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: They were also believed to have cross-reactivities with the S1 protein of SARS-CoV-2.

    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: They were also believed to have cross-reactivities with the S1 protein of SARS-CoV-2.

    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: They were also believed to have cross-reactivities with the S1 protein of SARS-CoV-2.

    Techniques: Chemiluminescent ELISA