sars cov 2 rbd protein  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD rFc Recombinant Protein COVID 19 Spike RBD Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of rabbit IgG1 at the C terminus
    Catalog Number:
    40592-V31H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
    		Buy from Supplier

    Structured Review

    Sino Biological sars cov 2 rbd protein
    A] Cell viability after incubation of Vero cells with imatinib for either 1 or 8 hours. and B] <t>SARS-CoV-2</t> neutralization profile post 1- and 8-hours exposure to imatinib. Inhibition of VSV pseudoparticles for SARS-CoV, SARS-CoV-2, MERS-CoV and VSV(control) after incubation with imatinib in C] Vero cells and D] Vero-TMPRSS2 cells. The red arrow indicates the concentration where no toxicity was observed microscopically anymore (15 nM). E] The association and dissociation curves obtained by BLI reflecting the binding of imatinib (0.78 to 6.25 µM) to immobilized SARS-CoV-2 RBD protein. Data fitted using the 1:1 binding model are shown in black.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein RBD YP 009724390 1 Arg319 Phe541 was expressed with the Fc region of rabbit IgG1 at the C terminus
    https://www.bioz.com/result/sars cov 2 rbd protein/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 rbd protein - by Bioz Stars, 2021-04
    94/100 stars

    Images

    1) Product Images from "Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19"

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.18.158196

    A] Cell viability after incubation of Vero cells with imatinib for either 1 or 8 hours. and B] SARS-CoV-2 neutralization profile post 1- and 8-hours exposure to imatinib. Inhibition of VSV pseudoparticles for SARS-CoV, SARS-CoV-2, MERS-CoV and VSV(control) after incubation with imatinib in C] Vero cells and D] Vero-TMPRSS2 cells. The red arrow indicates the concentration where no toxicity was observed microscopically anymore (15 nM). E] The association and dissociation curves obtained by BLI reflecting the binding of imatinib (0.78 to 6.25 µM) to immobilized SARS-CoV-2 RBD protein. Data fitted using the 1:1 binding model are shown in black.
    Figure Legend Snippet: A] Cell viability after incubation of Vero cells with imatinib for either 1 or 8 hours. and B] SARS-CoV-2 neutralization profile post 1- and 8-hours exposure to imatinib. Inhibition of VSV pseudoparticles for SARS-CoV, SARS-CoV-2, MERS-CoV and VSV(control) after incubation with imatinib in C] Vero cells and D] Vero-TMPRSS2 cells. The red arrow indicates the concentration where no toxicity was observed microscopically anymore (15 nM). E] The association and dissociation curves obtained by BLI reflecting the binding of imatinib (0.78 to 6.25 µM) to immobilized SARS-CoV-2 RBD protein. Data fitted using the 1:1 binding model are shown in black.

    Techniques Used: Incubation, Neutralization, Inhibition, Concentration Assay, Binding Assay

    A] Docked poses of the selected compounds at the receptor-binding domain of SARS-CoV-2 spike protein (inset: conformation of imatinib from molecular dynamics simulations showing important interactions with the receptor at the active site). B] MM-GBSA binding free energies for the selected compounds with negative control DMSO. Error bars indicate standard deviations for sampling from a whole simulation. Tyrosine kinase inhibitors ponatinib and imatinib displayed a high affinity to the RBD of the spike protein.
    Figure Legend Snippet: A] Docked poses of the selected compounds at the receptor-binding domain of SARS-CoV-2 spike protein (inset: conformation of imatinib from molecular dynamics simulations showing important interactions with the receptor at the active site). B] MM-GBSA binding free energies for the selected compounds with negative control DMSO. Error bars indicate standard deviations for sampling from a whole simulation. Tyrosine kinase inhibitors ponatinib and imatinib displayed a high affinity to the RBD of the spike protein.

    Techniques Used: Binding Assay, Negative Control, Sampling

    2) Product Images from "Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein"

    Article Title: Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.635677

    Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.
    Figure Legend Snippet: Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.
    Figure Legend Snippet: Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.

    Techniques Used: Purification, Filtration, SDS Page, Migration

    Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.
    Figure Legend Snippet: Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.
    Figure Legend Snippet: Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.

    Techniques Used: Stripping Membranes, Produced, Negative Control, Software

    3) Product Images from "SARS-CoV-2 neutralizing serum antibodies in cats: a serological investigation"

    Article Title: SARS-CoV-2 neutralizing serum antibodies in cats: a serological investigation

    Journal: bioRxiv

    doi: 10.1101/2020.04.01.021196

    ELISA of cat serum samples against the recombinant receptor binding domain (RBD) of SARS-CoV-2 spike. The dashed line is the positive cut-off. Each dot represents one individual sample within each antigen panel.
    Figure Legend Snippet: ELISA of cat serum samples against the recombinant receptor binding domain (RBD) of SARS-CoV-2 spike. The dashed line is the positive cut-off. Each dot represents one individual sample within each antigen panel.

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

    Virus neutralization test and western blotting assay of cat serum samples for SARS-CoV-2. (A) Morphology of SARS-CoV-2 viral plaques. Three representative sera are shown (#4, #14 and #15 corresponding to cat ID numbers in table 1 ) as well as hyperimmune sera of type I and II FIPV, and the virus input control. (B) Western blotting assay of cat or human serum samples for SARS-CoV-2. The convalescent serum of COVID-19 patient was used as a positive control. The negative cat serum of ELISA or healthy human serum was used as negative control. All of the detected serum samples were used at a dilution of 1:100. C-N, negative cat serum. H-P, human convalescent serum. H-N, healthy human serum. Red arrows, S protein. Blue arrows, N protein.
    Figure Legend Snippet: Virus neutralization test and western blotting assay of cat serum samples for SARS-CoV-2. (A) Morphology of SARS-CoV-2 viral plaques. Three representative sera are shown (#4, #14 and #15 corresponding to cat ID numbers in table 1 ) as well as hyperimmune sera of type I and II FIPV, and the virus input control. (B) Western blotting assay of cat or human serum samples for SARS-CoV-2. The convalescent serum of COVID-19 patient was used as a positive control. The negative cat serum of ELISA or healthy human serum was used as negative control. All of the detected serum samples were used at a dilution of 1:100. C-N, negative cat serum. H-P, human convalescent serum. H-N, healthy human serum. Red arrows, S protein. Blue arrows, N protein.

    Techniques Used: Neutralization, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    4) Product Images from "SARS-CoV-2 neutralizing serum antibodies in cats: a serological investigation"

    Article Title: SARS-CoV-2 neutralizing serum antibodies in cats: a serological investigation

    Journal: bioRxiv

    doi: 10.1101/2020.04.01.021196

    ELISA of cat serum samples against the recombinant receptor binding domain (RBD) of SARS-CoV-2 spike. The dashed line is the positive cut-off. Each dot represents one individual sample within each antigen panel.
    Figure Legend Snippet: ELISA of cat serum samples against the recombinant receptor binding domain (RBD) of SARS-CoV-2 spike. The dashed line is the positive cut-off. Each dot represents one individual sample within each antigen panel.

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

    Virus neutralization test and western blotting assay of cat serum samples for SARS-CoV-2. (A) Morphology of SARS-CoV-2 viral plaques. Three representative sera are shown (#4, #14 and #15 corresponding to cat ID numbers in table 1 ) as well as hyperimmune sera of type I and II FIPV, and the virus input control. (B) Western blotting assay of cat or human serum samples for SARS-CoV-2. The convalescent serum of COVID-19 patient was used as a positive control. The negative cat serum of ELISA or healthy human serum was used as negative control. All of the detected serum samples were used at a dilution of 1:100. C-N, negative cat serum. H-P, human convalescent serum. H-N, healthy human serum. Red arrows, S protein. Blue arrows, N protein.
    Figure Legend Snippet: Virus neutralization test and western blotting assay of cat serum samples for SARS-CoV-2. (A) Morphology of SARS-CoV-2 viral plaques. Three representative sera are shown (#4, #14 and #15 corresponding to cat ID numbers in table 1 ) as well as hyperimmune sera of type I and II FIPV, and the virus input control. (B) Western blotting assay of cat or human serum samples for SARS-CoV-2. The convalescent serum of COVID-19 patient was used as a positive control. The negative cat serum of ELISA or healthy human serum was used as negative control. All of the detected serum samples were used at a dilution of 1:100. C-N, negative cat serum. H-P, human convalescent serum. H-N, healthy human serum. Red arrows, S protein. Blue arrows, N protein.

    Techniques Used: Neutralization, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    5) Product Images from "Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein"

    Article Title: Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.635677

    Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.
    Figure Legend Snippet: Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.
    Figure Legend Snippet: Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.

    Techniques Used: Purification, Filtration, SDS Page, Migration

    Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.
    Figure Legend Snippet: Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.
    Figure Legend Snippet: Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.

    Techniques Used: Stripping Membranes, Produced, Negative Control, Software

    6) Product Images from "Rapid Development of SARS-CoV-2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates"

    Article Title: Rapid Development of SARS-CoV-2 Spike Protein Receptor-Binding Domain Self-Assembled Nanoparticle Vaccine Candidates

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c08379

    Neutralization activity of sera from mice immunized with RBD monomer or RBD-conjugated nanoparticles. (A) SARS-CoV-2 pseudovirus neutralization assay showing the NT 90 . (B) SARS-CoV-2 live virus neutralization assay showing the FRNT 90 . The statistical significance of the difference of neutralizing titers of mice immunized with immunogen combined with either AddaVax or SAS as the adjuvant was calculated using the unpaired two-tailed nonparametric Mann–Whitney U test. * p
    Figure Legend Snippet: Neutralization activity of sera from mice immunized with RBD monomer or RBD-conjugated nanoparticles. (A) SARS-CoV-2 pseudovirus neutralization assay showing the NT 90 . (B) SARS-CoV-2 live virus neutralization assay showing the FRNT 90 . The statistical significance of the difference of neutralizing titers of mice immunized with immunogen combined with either AddaVax or SAS as the adjuvant was calculated using the unpaired two-tailed nonparametric Mann–Whitney U test. * p

    Techniques Used: Neutralization, Activity Assay, Mouse Assay, Two Tailed Test, MANN-WHITNEY

    7) Product Images from "Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein"

    Article Title: Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.635677

    Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.
    Figure Legend Snippet: Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.
    Figure Legend Snippet: Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.

    Techniques Used: Purification, Filtration, SDS Page, Migration

    Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.
    Figure Legend Snippet: Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.
    Figure Legend Snippet: Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.

    Techniques Used: Stripping Membranes, Produced, Negative Control, Software

    8) Product Images from "A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2"

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21037-2

    Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Injection, Infection, Enzyme-linked Immunospot, Two Tailed Test

    Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Transgenic Assay, Two Tailed Test

    Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Transgenic Assay, Mouse Assay, Histopathology, Infection, Immunohistochemistry, Two Tailed Test, Injection

    Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.
    Figure Legend Snippet: Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Transfection, Expressing, Western Blot, Electron Microscopy

    9) Product Images from "Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2"

    Article Title: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.02.27.433054

    The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).
    Figure Legend Snippet: The recombinant SARS-CoV-2 HexaPro spike protein. (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from S-2P construct are indicated by the red arrows shown below the construct. (B) The HexaPro protein expressed in HEK293T cells was purified and characterized by SDS-PAGE (left), western blot using a commercial anti-RBD (middle), and western blot using pooled convalescence sera (right).

    Techniques Used: Recombinant, Construct, Purification, SDS Page, Western Blot

    10) Product Images from "A serological survey of SARS-CoV-2 in cat in Wuhan"

    Article Title: A serological survey of SARS-CoV-2 in cat in Wuhan

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1817796

    Virus neutralization test and Western blot assay of cat serum samples for SARS-CoV-2 (A) Cat#14, Cat#15 and Cat#4 sera were 3-fold serially diluted and mixed with SARS-CoV-2; after incubated at 37°C for 1 h, the mixture was used to infect Vero E6 cells, and replaced with semi-solid media 1 h later. The plates were fixed and stained 3 days later. All samples were tested in duplicate. (B) Western blot of purified SARS-CoV-2 with cat or human sera. All sera were diluted 100 folds. C-N, negative cat serum. H-P, human convalescent serum. H-N, healthy human serum.
    Figure Legend Snippet: Virus neutralization test and Western blot assay of cat serum samples for SARS-CoV-2 (A) Cat#14, Cat#15 and Cat#4 sera were 3-fold serially diluted and mixed with SARS-CoV-2; after incubated at 37°C for 1 h, the mixture was used to infect Vero E6 cells, and replaced with semi-solid media 1 h later. The plates were fixed and stained 3 days later. All samples were tested in duplicate. (B) Western blot of purified SARS-CoV-2 with cat or human sera. All sera were diluted 100 folds. C-N, negative cat serum. H-P, human convalescent serum. H-N, healthy human serum.

    Techniques Used: Neutralization, Western Blot, Incubation, Staining, Purification

    ELISA of cat serum samples against the recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. The dashed line is the cut-off. Each dot represents one individual sample within each antigen panel. Before, serum samples collected between Mar. and May, 2019 prior to COVID-19 outbreak; After, serum samples collected between Jan to Mar., 2020 after COVID-19 outbreak; FIPV- I, hyperimmune sera against type I feline infectious peritonitis virus (FIPV); FIPV-II, hyperimmune sera against type II FIPV.
    Figure Legend Snippet: ELISA of cat serum samples against the recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. The dashed line is the cut-off. Each dot represents one individual sample within each antigen panel. Before, serum samples collected between Mar. and May, 2019 prior to COVID-19 outbreak; After, serum samples collected between Jan to Mar., 2020 after COVID-19 outbreak; FIPV- I, hyperimmune sera against type I feline infectious peritonitis virus (FIPV); FIPV-II, hyperimmune sera against type II FIPV.

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

    The dynamic change of cat serum antibody for SARS-CoV-2. (A) ELISA detection and (B) neutralization test of cat serums. The serums of Cat#14 and Cat#15 were collected every 10 days from Mar. 3 to Jul. 11. Then the ELISA against SARS-CoV-2 RBD and the virus neutralization test were performed.
    Figure Legend Snippet: The dynamic change of cat serum antibody for SARS-CoV-2. (A) ELISA detection and (B) neutralization test of cat serums. The serums of Cat#14 and Cat#15 were collected every 10 days from Mar. 3 to Jul. 11. Then the ELISA against SARS-CoV-2 RBD and the virus neutralization test were performed.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Neutralization

    11) Product Images from "Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in hamsters"

    Article Title: Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in hamsters

    Journal: bioRxiv

    doi: 10.1101/2021.03.08.433449

    HumVHH_S56A/LALA-Fc/Gen2 neutralizes SARS-CoV-2 variants of concern. a . Surface view of SARS-CoV-2 RBD (grey) with VHH72 (green cartoon, bottom) and the N-terminal helixes of ACE2 (blue cartoon, top). The RBD-residues K417, N439, E484 and N501 (orange) are indicated. b. Binding of humVHH_S56A/LALA-Fc/Gen2 (left), CB6 (middle) and palivizumab (right) to SARS-CoV-1 spike with the RBD replaced by WT, N439K, K417N, E484K, N501Y or (K417N + E484K + N501Y) RBD of SARS-CoV-2, expressed on the surface of 293T cells. Data points represent the ratio of the mean fluorescence intensity (MFI) of transfected (GFP + ) cells over the MFI of non-transfected (GFP - ) cells, as determined by flow cytometry. c . SARS-CoV-2 plaque reduction neutralization assay with 3 fold serial dilutions of the indicated VHH-Fc fusion constructs using BetaCov/Belgium/GHB-03021/2020, B1.1.7, or B.1.351 variant viruses.
    Figure Legend Snippet: HumVHH_S56A/LALA-Fc/Gen2 neutralizes SARS-CoV-2 variants of concern. a . Surface view of SARS-CoV-2 RBD (grey) with VHH72 (green cartoon, bottom) and the N-terminal helixes of ACE2 (blue cartoon, top). The RBD-residues K417, N439, E484 and N501 (orange) are indicated. b. Binding of humVHH_S56A/LALA-Fc/Gen2 (left), CB6 (middle) and palivizumab (right) to SARS-CoV-1 spike with the RBD replaced by WT, N439K, K417N, E484K, N501Y or (K417N + E484K + N501Y) RBD of SARS-CoV-2, expressed on the surface of 293T cells. Data points represent the ratio of the mean fluorescence intensity (MFI) of transfected (GFP + ) cells over the MFI of non-transfected (GFP - ) cells, as determined by flow cytometry. c . SARS-CoV-2 plaque reduction neutralization assay with 3 fold serial dilutions of the indicated VHH-Fc fusion constructs using BetaCov/Belgium/GHB-03021/2020, B1.1.7, or B.1.351 variant viruses.

    Techniques Used: Binding Assay, Fluorescence, Transfection, Flow Cytometry, Neutralization, Construct, Variant Assay

    Therapeutic administration of VHH72-Fc constructs restricts SARS-CoV-2 virus replication in Syrian hamsters. a-c. Hamsters were challenged with 1×10 4 PFU of BetaCoV/Munich/BavPat1/2020 and 4 hours later injected intraperitoneally with 20, 7 or 2 mg/kg of bivalent humVHH_S56A/LALAPG-Fc/Gen2 or tetravalent (humVHH_S56A) 2 /LALAPG-Fc/Gen2. The negative control group was treated with 20 mg/kg of palivizumab, injected 4 hours after the challenge infection; hamsters in a prophylactic control group received 20 mg/kg of humVHH_S56A/LALAPG-Fc/Gen2 one day before the challenge. ( a ) Lung virus loads, ( b ) lung viral RNA copies, and ( c ) gross lung pathology determined on day 4 after infection. d,e . Hamsters received an intraperitoneal injection of 7 mg/kg of humVHH_S56A/LALAPG-Fc/Gen2 one day prior to challenge or were treated by intraperitoneal injection of 1 or 7 mg/kg of humVHH_S56A/LALAPG-Fc/Gen2 or (humVHH_S56A) 2 /LALAPG-Fc/Gen2 19h after infection with 2×10 6 PFU of passage 6 BetaCov/Belgium/GHB-03021/2020. Seven mg/kg of palivizumab was used as a negative control treatment. ( d ) Virus load and ( e ) viral RNA levels in the lungs on day 4 after challenge. f,g . Hamsters were treated with 4mg/kg of palivizumab, humVHH_S56A/LALA-Fc/Gen2 or humVHH/LALA-Fc/Gen2 injected intraperitoneally 24h after challenge with 2×10 6 PFU of passage 6 BetaCov/Belgium/GHB-03021/2020. Viral RNA and infectious virus were determined in lung tissue on day 4 after infection. Data were analyzed with the Mann-Whitney U-test using GraphPad Prism software. *, p
    Figure Legend Snippet: Therapeutic administration of VHH72-Fc constructs restricts SARS-CoV-2 virus replication in Syrian hamsters. a-c. Hamsters were challenged with 1×10 4 PFU of BetaCoV/Munich/BavPat1/2020 and 4 hours later injected intraperitoneally with 20, 7 or 2 mg/kg of bivalent humVHH_S56A/LALAPG-Fc/Gen2 or tetravalent (humVHH_S56A) 2 /LALAPG-Fc/Gen2. The negative control group was treated with 20 mg/kg of palivizumab, injected 4 hours after the challenge infection; hamsters in a prophylactic control group received 20 mg/kg of humVHH_S56A/LALAPG-Fc/Gen2 one day before the challenge. ( a ) Lung virus loads, ( b ) lung viral RNA copies, and ( c ) gross lung pathology determined on day 4 after infection. d,e . Hamsters received an intraperitoneal injection of 7 mg/kg of humVHH_S56A/LALAPG-Fc/Gen2 one day prior to challenge or were treated by intraperitoneal injection of 1 or 7 mg/kg of humVHH_S56A/LALAPG-Fc/Gen2 or (humVHH_S56A) 2 /LALAPG-Fc/Gen2 19h after infection with 2×10 6 PFU of passage 6 BetaCov/Belgium/GHB-03021/2020. Seven mg/kg of palivizumab was used as a negative control treatment. ( d ) Virus load and ( e ) viral RNA levels in the lungs on day 4 after challenge. f,g . Hamsters were treated with 4mg/kg of palivizumab, humVHH_S56A/LALA-Fc/Gen2 or humVHH/LALA-Fc/Gen2 injected intraperitoneally 24h after challenge with 2×10 6 PFU of passage 6 BetaCov/Belgium/GHB-03021/2020. Viral RNA and infectious virus were determined in lung tissue on day 4 after infection. Data were analyzed with the Mann-Whitney U-test using GraphPad Prism software. *, p

    Techniques Used: Construct, Injection, Negative Control, Infection, MANN-WHITNEY, Software

    Enhanced affinity and neutralizing activity of computationally predicted VHH72 variant. a-c. Prototype WT-VHH/12GS-WT-Fc protects K18-hACE2 transgenic mice against SARS-CoV-2 challenge. Mice were injected intraperitoneally with 5 mg/kg of either WT-VHH/12GS-WT-Fc (n = 17) or control M2e-VHH-Fc (directed against the matrix protein 2 ectodomain of influenza A; n = 10). Seven hours later, the mice were challenged with 8 x 10 3 PFU of a clinical SARS-CoV-2 isolate. ( a ) Body weight (symbols represent means ± SD, p
    Figure Legend Snippet: Enhanced affinity and neutralizing activity of computationally predicted VHH72 variant. a-c. Prototype WT-VHH/12GS-WT-Fc protects K18-hACE2 transgenic mice against SARS-CoV-2 challenge. Mice were injected intraperitoneally with 5 mg/kg of either WT-VHH/12GS-WT-Fc (n = 17) or control M2e-VHH-Fc (directed against the matrix protein 2 ectodomain of influenza A; n = 10). Seven hours later, the mice were challenged with 8 x 10 3 PFU of a clinical SARS-CoV-2 isolate. ( a ) Body weight (symbols represent means ± SD, p

    Techniques Used: Activity Assay, Variant Assay, Transgenic Assay, Mouse Assay, Injection

    VHH72_S56A-Fc constructs have increased affinity and SARS-CoV-2 neutralizing activity. a. Binding affinity of VHH72-Fc variants to immobilized mouse Fc-fused SARS-CoV-2 RBD (RBD-mFc). Apparent kinetics of the 2:2 interaction is based on a global 1:1 fit of the replicate (n = 2) data; values are the averages of replicates. b. Binding of the indicated VHH72-Fc constructs (see Supplementary Table 2 for a description) to coated SARS-CoV-2 spike determined by ELISA (data points are mean ± SD; n=3). c. Binding of the indicated VHH72-Fc constructs to cell surface expressed SARS-CoV-2 spike determined by flow cytometry. The graph shows the mean (n=2) ratio of the MFI of transfected (GFP + ) cells over the MFI of non-transfected (GFP - ) cells. d. Inhibition of ACE-2/RBD interaction determined by AlphaLISA (amplified luminescent proximity homogeneous assay). Biotinylated SARS-CoV-2 RBD was loaded on streptavidin coated Alpha Donor beads and human ACE-2-mFc protein was captured on anti-mouse IgG acceptor beads. Interference of the donor-acceptor bead interaction was assessed for serial dilutions of the indicated VHH-Fc constructs. Graph pad Prism was used for curve fitting and IC 50 determination of triplicate measurements. e . Dose-dependent inhibition of SARS-CoV-2 RBD binding to the surface of VeroE6 cells in the presence of the indicated VHH72-Fc constructs as determined by flow cytometry. The graph shows the mean (n=2 ± SD) percentage of cells that bind RBD. f. A SARS-CoV-2 plaque reduction neutralization assay was performed with 3 fold serial dilutions of the indicated VHH-Fc fusion constructs. Thirtysix hours after infection, the cells fixed with 3.7% paraformaldehyde and stained with 0.5% crystal violet. Data points in the graph represent the number of plaques and are representative of one experiment that was repeated once. g-h . humVHH_S56A/LALAPG-Fc and (humVHH) 2 /WT-Fc at 20 mg/kg protect hamsters against SARS-CoV-2 challenge. Hamsters were intraperitoneally injected with 20 mg/kg of palivizumab, humVHH_S56A/LALAPG-Fc or (humVHH) 2 /WT-Fc and challenged the next day with 2×10 6 PFU of passage 6 BetaCov/Belgium/GHB-03021/2020. ( g ). Infectious virus in lungs and ( h ) viral RNA in lungs, ileum and stool determined on day 4 after the challenge. ( i ) Severity score of dilated bronchi on day 4 after the challenge.
    Figure Legend Snippet: VHH72_S56A-Fc constructs have increased affinity and SARS-CoV-2 neutralizing activity. a. Binding affinity of VHH72-Fc variants to immobilized mouse Fc-fused SARS-CoV-2 RBD (RBD-mFc). Apparent kinetics of the 2:2 interaction is based on a global 1:1 fit of the replicate (n = 2) data; values are the averages of replicates. b. Binding of the indicated VHH72-Fc constructs (see Supplementary Table 2 for a description) to coated SARS-CoV-2 spike determined by ELISA (data points are mean ± SD; n=3). c. Binding of the indicated VHH72-Fc constructs to cell surface expressed SARS-CoV-2 spike determined by flow cytometry. The graph shows the mean (n=2) ratio of the MFI of transfected (GFP + ) cells over the MFI of non-transfected (GFP - ) cells. d. Inhibition of ACE-2/RBD interaction determined by AlphaLISA (amplified luminescent proximity homogeneous assay). Biotinylated SARS-CoV-2 RBD was loaded on streptavidin coated Alpha Donor beads and human ACE-2-mFc protein was captured on anti-mouse IgG acceptor beads. Interference of the donor-acceptor bead interaction was assessed for serial dilutions of the indicated VHH-Fc constructs. Graph pad Prism was used for curve fitting and IC 50 determination of triplicate measurements. e . Dose-dependent inhibition of SARS-CoV-2 RBD binding to the surface of VeroE6 cells in the presence of the indicated VHH72-Fc constructs as determined by flow cytometry. The graph shows the mean (n=2 ± SD) percentage of cells that bind RBD. f. A SARS-CoV-2 plaque reduction neutralization assay was performed with 3 fold serial dilutions of the indicated VHH-Fc fusion constructs. Thirtysix hours after infection, the cells fixed with 3.7% paraformaldehyde and stained with 0.5% crystal violet. Data points in the graph represent the number of plaques and are representative of one experiment that was repeated once. g-h . humVHH_S56A/LALAPG-Fc and (humVHH) 2 /WT-Fc at 20 mg/kg protect hamsters against SARS-CoV-2 challenge. Hamsters were intraperitoneally injected with 20 mg/kg of palivizumab, humVHH_S56A/LALAPG-Fc or (humVHH) 2 /WT-Fc and challenged the next day with 2×10 6 PFU of passage 6 BetaCov/Belgium/GHB-03021/2020. ( g ). Infectious virus in lungs and ( h ) viral RNA in lungs, ileum and stool determined on day 4 after the challenge. ( i ) Severity score of dilated bronchi on day 4 after the challenge.

    Techniques Used: Construct, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Transfection, Inhibition, Amplification, Neutralization, Infection, Staining, Injection

    12) Product Images from "A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2"

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21037-2

    Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Injection, Infection, Enzyme-linked Immunospot, Two Tailed Test

    Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Mouse Assay, Transgenic Assay, Two Tailed Test

    Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Transgenic Assay, Mouse Assay, Histopathology, Infection, Immunohistochemistry, Two Tailed Test, Injection

    Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.
    Figure Legend Snippet: Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

    Techniques Used: Binding Assay, Transfection, Expressing, Western Blot, Electron Microscopy

    13) Product Images from "Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein"

    Article Title: Development of a Colloidal Gold-Based Immunochromatographic Strip for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.635677

    Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.
    Figure Legend Snippet: Stability evaluation of the strip. The sensitivities of fresh strips (left) and strips after 6 months of storage (right) were determined. 1–9: The SARS-CoV-2 RBD protein produced in this study diluted ranging from 4,000 to 62.5 ng/mL by two times ratio; N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.
    Figure Legend Snippet: Purification of SARS-CoV-2 RBD protein. Analytical gel filtration profile of SARS-CoV-2 RBD protein with HisTrap TM excel. The 280-nm absorbance curve was shown. SDS-PAGE migration profiles of the sample purified was shown.

    Techniques Used: Purification, Filtration, SDS Page, Migration

    Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.
    Figure Legend Snippet: Specificity evaluation of the strip. 1: SARS-CoV-2 RBD protein produced in this study; 2: SARS-CoV-2 S1 protein (Sino Biological Inc.); 3: SARS-CoV S1 protein (Sino Biological Inc.); 4: MERS-CoV S1 protein (Sino Biological Inc.); 5: IBV-S protein (Shandong Lvdu Bio-technique Industry); 6: PEDV-S protein (Shandong Lvdu Bio-technique Industry); 7: A/Swine/Guangxi/NN1994/2013 (H1N1); 8: A/Swine/Guangxi/NNXD/2016 (H3N2); 9: A/Duck/Yunnan/YN-9/2016 (H5N6); 10: A/Chicken/Huizhou/HZ-3/2016 (H7N9); 11: A/Chicken/Guangdong/V/2008 (H9N2); N: PBS negative control.

    Techniques Used: Stripping Membranes, Produced, Negative Control

    Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.
    Figure Legend Snippet: Sensitivity evaluation of the strip. (A) SARS-CoV-2 RBD protein produced in this study, 1–11: diluted positive sample ranging from 4,000 to 15.63 ng/mL by two times ratio, N: PBS negative control. (B) SARS-CoV-2 S1 protein (Sino Biological Inc.); 1–9: diluted positive sample ranging from 4,000 to 62.5 ng/mL by two times ratio, N: PBS negative control. (C,D) The colored membranes of SARS-CoV-2 RBD protein produced in this study and SARS-CoV-2 S1 protein (Sino Biological Inc.) were screened under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed by AIS software.

    Techniques Used: Stripping Membranes, Produced, Negative Control, Software

    Related Articles

    Infection:

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    Article Snippet: .. SARS-CoV-2 is highly transmissible during both the pre-symptomatic and acute symptomatic phases and the infection fatality rate has been reported as high as 3.4% . .. COVID-19 often develops into severe illness, including pneumonia.

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: .. To evaluate whether these 6 antibodies can recognize SARS-CoV-2 in FFPE specimens, we performed IHC on FFPE pellets of Vero 76 cells infected with SARS-CoV-2. .. We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV NP (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets ( ).

    other:

    Article Title: Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19
    Article Snippet: The plated concentrations of ACE2 receptor were 1.0 μg/mL for SARS-CoV-2 RBD and 2.0 μg/mL for SARS-CoV-2 S1.

    Immunohistochemistry:

    Article Title: Placental SARS-CoV-2 in a patient with mild COVID-19 disease
    Article Snippet: Immunohistochemistry (IHC) staining of SARS-CoV-2 virus in a COVID-19 negative patient, delivery prior to the COVID-19 outbreak (Figure 2a). .. IHC of SARS-CoV-2 from three placental sections (2b: under umbilical cord, 2c: central placental disc, 2d-e: peripheral placental disc at 20x and 40x). .. IHC of cytokeratin-7 (CK-7) marker in control ferret nasal turbinate tissue (Figure 2f).

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: .. To evaluate whether these 6 antibodies can recognize SARS-CoV-2 in FFPE specimens, we performed IHC on FFPE pellets of Vero 76 cells infected with SARS-CoV-2. .. We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV NP (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets ( ).

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: We also identify 2 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S and RNA in the same FFPE section. .. Identification of antibodies suitable for detection of SARS-CoV-2 by IHC and IFA in FFPE specimens.). ..

    Titration:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Microneutralization Assay:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Expressing:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Cell Culture:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Concentration Assay:

    Article Title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
    Article Snippet: The dose titer of PsV was determined by infecting ACE-2 and TMPRSS2 expressing 293T cells for 48 h and using Celigo imaging system for imaging and counting virus infected fluorescent cells . .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media. .. Neutralizing Antibody (Nab) Titration Assay for SARS-CoV-2 For microneutralization assay, ACE2-TMPRSS2 expressing 293 T cells were cultured overnight in a half area 96-well plate compatible with Nexcelom Celigo imager at a concentration of 1 ×104 cells per well in 100 µl of complete media.

    Binding Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: The number of infected cells per well were counted using the ImageQuant TL software. .. BLIThe binding kinetics of imatinib on SARS-CoV-2 RBD protein were studied using a BLItz® system (FortéBio). .. Experiments were conducted using the advanced kinetics mode, at room temperature and a buffer system consisting of 1X Kinetics Buffer (FortéBio), 5% anhydrous dimethyl sulfoxide (DMSO; Sigma Aldrich).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: .. To evaluate whether these 6 antibodies can recognize SARS-CoV-2 in FFPE specimens, we performed IHC on FFPE pellets of Vero 76 cells infected with SARS-CoV-2. .. We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV NP (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets ( ).

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: We also identify 2 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S and RNA in the same FFPE section. .. Identification of antibodies suitable for detection of SARS-CoV-2 by IHC and IFA in FFPE specimens.). ..

    Immunofluorescence:

    Article Title: Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
    Article Snippet: We also identify 2 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S and RNA in the same FFPE section. .. Identification of antibodies suitable for detection of SARS-CoV-2 by IHC and IFA in FFPE specimens.). ..

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    • sars cov 2 spike protein  (Sino Biological)
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    Sino Biological sars cov 2 spike protein
    A replication-competent <t>VSV/SARS-CoV-2</t> chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.
    Sars Cov 2 Spike Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 2 rbd  (Sino Biological)
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    Sino Biological sars cov 2 rbd
    RU169 output clone diversity Using the <t>SARS-CoV-2</t> RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.
    Sars Cov 2 Rbd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sars cov 2 2019 ncov spike rbd antibody rabbit pab  (Sino Biological)
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    Sino Biological sars cov 2 2019 ncov spike rbd antibody rabbit pab
    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates <t>SARS-CoV-2</t> PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.
    Sars Cov 2 2019 Ncov Spike Rbd Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Infection, Purification, Standard Deviation

    Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Infection, Flow Cytometry, Luciferase, Activity Assay

    Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Activity Assay, Incubation, Infection, Immunostaining, Luciferase

    Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Infection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Selection, Clone Assay, Isolation, Flow Cytometry, Staining, Generated, Luciferase, Activity Assay, Standard Deviation

    Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Neutralization, Incubation, Infection, Immunostaining, Luciferase

    Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Journal: bioRxiv

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    doi: 10.1101/2020.06.08.140871

    Figure Lengend Snippet: Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Article Snippet: To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ).

    Techniques: Plasmid Preparation, Modification, Luciferase, Generated, Cotransfection, Construct, Infection, Activity Assay, Standard Deviation, Clone Assay

    RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: RU169 output clone diversity Using the SARS-CoV-2 RBD as the target of library panning and FACS selection for screen RU169 produced a high number of unique clones, indicating high, unexplored, diversity in the output.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: FACS, Selection, Produced, Clone Assay

    BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: BLI kinetics of selected scFv clones from the RU169 RBD screen. scFv were cloned into an AviTag™ biotinylation vector, as described in the Materials and Methods, expressed and purified by Ni-NTA resin. scFv were loaded onto a streptavidin BLI sensor and the association/dissociation kinetics of binding to soluble SARS-CoV-2 S1 trimer (100 nM) were measured using BLI. The K D of the scFvs for the S1 target ranged from 1 nM to 400 nM.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Clone Assay, Plasmid Preparation, Purification, Binding Assay

    Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: Anti-RBD clones in IgG1 format form long-lived complexes with SARS-CoV-2 S1 trimer and potently inhibit the interaction with ACE2 in vitro . A. Dissociation kinetics of IgG1 anti-RBD clones from SARS-CoV-2 S1 trimer. Biotinylated SARS-CoV-2 S1 trimer was bound to a streptavidin BLI sensor. IgG1 anti-RBD clones were bound (100 nM) and the dissociation followed for 4 hours in PBS at 25°C. B. ACE2-S1 Dynabead assay with molar equivalents of mAb clones to S1 trimer.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Clone Assay, In Vitro

    FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: FACS strategy of screen RU167 for scFv inhibiting the SARS-CoV-2 RBD/ACE2 interaction The FACS-based screening strategy for screen RU167 to isolate antibodies that bound SARS-CoV-2 RBD and specifically inhibited co-binding of RBD to the human ACE2 protein. The viral RBD and the ACE2 protein were labeled with different fluorophores (A). Binding to cells expressing scFv clones that bound RBD and blocking the ACE2-binding site (B) would be observed and gated positively for in the FACS plot for events which were RBD-dye HIGH and ACE2-dye LOW (C).

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: FACS, Binding Assay, Labeling, Expressing, Clone Assay, Blocking Assay

    BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: BLI kinetics of anti-RBD diabodies AviTag™ biotinylated SARS-CoV-2 S1 trimer was loaded onto a BLI sensor and the association/dissociation kinetics of binding to anti-RBD diabodies (100 nM) were measured using BLI. The K D s of the dbs to the S1 target ranged from 84 pM to 1 nM.

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Binding Assay

    Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Journal: bioRxiv

    Article Title: Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

    doi: 10.1101/2020.07.27.224089

    Figure Lengend Snippet: Cytometry plots of ACE2-S1 Dynabead assay of anti-RBD diabodies The degree of inhibition of the ACE2 and SARS-CoV-2 S1 trimer interaction by stoichiometric amounts of anti-RBD diabodies was determined using a Dynabead assay as described in the Materials and Methods. The degree of bead fluorescence was indicative of the amount of dye-labeled S1 trimer that was bound to ACE2. Inhibition of the interaction by anti-RBD diabodies resulted in a reduction in fluorescence. The first panel is the SSC/FSC indicating the P1 gating of beads. The second panel is the biotin-blocked control (no ACE2/S1 interaction) and the third panel is the no anti-RBD control (maximum ACE2/S1 interaction. Each subsequent row represents a db clone at 1:1, 5:1 and 10:1 stoichiometric ratios to the soluble SARS-CoV-2 S1 trimer. The data are summarized graphically in Figure 3 .

    Article Snippet: ACE2-S1 inhibition assayThe ability of RBD-binding antibodies to block the high-affinity interaction between SARS-CoV-2 RBD and human ACE2 protein was tested in a bead-binding assay.

    Techniques: Cytometry, Inhibition, Fluorescence, Labeling

    ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: ELISA ( x -axis) vs. LFRET ( y -axis) results by disease severity. ( a ) Anti-NP IgA ELISA vs. anti-NP LFRET (N = 81, R = 0.25). ( b ) anti-NP IgG ELISA vs. anti-NP LFRET (N = 129, R = 0.62). ( c ) anti-NP IgM ELISA vs. anti-NP LFRET (N = 81, R = 0.13). ( d ) anti-SP IgA ELISA vs. anti-SP LFRET (N = 129, R = 0.53). ( e ) anti-SP IgG ELISA vs. anti-SP LFRET (N = 129, R = 0.62). ( f ) anti-SP IgM ELISA vs. anti-SP LFRET (N = 81, R = 0.56). Color of the dot indicates SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal and vertical black lines indicate LFRET and ELISA cutoffs. On the x -axis, ELISA absorbance on a logarithmic scale and on the y -axis, LFRET signal on a logarithmic scale. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. R = Pearson’s correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Microneutralization vs. LFRET and ELISA. Microneutralization titers are on the x -axis and LFRET signal or ELISA absorbance on the y -axis. Logarithmic scale is used on both axes. ( a ) Microneutralization titer vs. anti-SP LFRET signal (N = 107, ρ = 0.87). ( b – d ) Microneutralization titer vs. anti-SP IgG, IgA and IgM ELISA (N = 107, 107 and 67, ρ = 0.68, 0.86 and 0.81). ( e ) Microneutralization titer vs. anti-NP LFRET signal (N = 107, ρ = 0.83). ( f – h ) Microneutralization titer vs. anti-NP IgG, IgA and IgM ELISA (N = 107, 67 and 67, ρ = 0.81, 0.69 and 0.61). Color of the dots indicate SARS-CoV-2 PCR result and disease severity: cyan = PCR negative; yellow = non-hospitalized, PCR-positive; red = non-ICU hospitalized, PCR positive; black = hospitalized in ICU, PCR positive. Horizontal black lines indicate LFRET/ELISA cutoffs. SP = spike glycoprotein. NP = nucleoprotein. LFRET = protein L–based time-resolved Förster resonance energy transfer immunoassay. ELISA = enzyme immunoassay. ρ = Spearman’s rank correlation coefficient.

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Förster Resonance Energy Transfer

    Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Journal: Viruses

    Article Title: A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

    doi: 10.3390/v13020143

    Figure Lengend Snippet: Simplified protocol for SARS-CoV-2 NP and SP LFRET assay. Eu-NP/-SP = Europium-labeled nucleoprotein/spike glycoprotein. AF-L = Alexa Fluor™ 647 -labeled protein L. TR-FRET = time-resolved Förster resonance energy transfer. RT = room temperature. TBS+BSA (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.2% BSA) was used for all dilutions. On-plate dilutions were 5 nM Eu-NP/500 nM AF-L/serum 1/25 for anti-NP and 5 nM Eu-SP/250 nM AF-L/serum 1/100 for anti-SP LFRET. For further details see the prior publication [ 5 ].

    Article Snippet: At 48 h, the medium was analyzed for the presence of SARS-CoV-2 SP by dot blotting; briefly via drying 2.5 µL of the supernatant onto a nitrocellulose membrane, which then was blocked (3% skim milk in Tris-buffered saline with 0.05% Tween-20), washed, probed with rabbit anti-RBD (40592-T62, Sino Biological, Beijing, China), washed, probed with anti-rabbit IRDye800 (LI-COR Biosciences, Lincoln, NE, USA), washed, and read using Odyssey Infrared Imaging System (LI-COR Biosciences).

    Techniques: Labeling, Förster Resonance Energy Transfer