sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research  (Sino Biological)


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    Sino Biological sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research
    Detection of <t>SARS-CoV-2</t> antibodies in human saliva. (a) A confocal fluorescence image of IgG signals in the saliva of 4 recovered COVID-19 patients (denoted as P1-P4) and 11 healthy controls (denoted as P5-P15) and a 10 4 times diluted serum of a PCR-confirmed COVID-19 patient as a reference (denoted as ‘Ref’). Saliva was collected by a simple spitting method as shown in the schematic. (b) Median fluorescence intensity (MFI) signals of anti-S1 and anti-RBD IgG measured in the saliva samples and PCR-positive COVID-19 serum reference with background signals subtracted. The error bars indicate one standard deviation away from the mean.
    Sars Cov 2 2019 Ncov Spike Rbd His Recombinant Protein Covid 19 Spike Rbd Research, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research/product/Sino Biological
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    sars cov 2 2019 ncov spike rbd his recombinant protein covid 19 spike rbd research - by Bioz Stars, 2021-04
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    Images

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

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

    Journal: bioRxiv

    doi: 10.1101/2020.06.16.155580

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

    Techniques Used: Fluorescence, Polymerase Chain Reaction, Standard Deviation

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

    Techniques Used: Polymerase Chain Reaction, Fluorescence, Standard Deviation

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

    Techniques Used: Polymerase Chain Reaction, Labeling

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

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

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

    Techniques Used: Polymerase Chain Reaction

    2) Product Images from "Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity"

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

    Journal: JCI Insight

    doi: 10.1172/jci.insight.142386

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

    Techniques Used: Enzyme-linked Immunosorbent Assay

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

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

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

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

    Journal: Biosensors & Bioelectronics

    doi: 10.1016/j.bios.2020.112572

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

    Techniques Used: Chemiluminescent ELISA

    Related Articles

    Recombinant:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: 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). .. Recombinant His-tagged SARS-CoV-2 RBD protein (40592-V08H; Sino Biological) at a concentration of 10 µg/ml was loaded on Anti-Penta-HIS (HIS1K) Biosensors (FortéBio), followed by a washing step with assay buffer to block the unoccupied sensor surface. .. The association and dissociation profiles of imatinib (Sigma Aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µM to 0.78 µM).

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: The normal human serum (H4522-20ML), which was used as the dilution buffer and as one of the negative controls in IgG detection experiments, and the heat-inactivated normal human serum (H5667-20ML), which was used as another negative control in IgG detection experiments, were both purchased from Millipore Sigma. .. Human-cell-expressed SARS-CoV-2 Spike S1-His recombinant protein (40591-V08H), human-cell-expressed SARS-CoV-2 Spike RBD-His recombinant protein (40592-V08H) and insect-cell-expressed SARS-CoV Spike S1-His recombinant protein (40150-V08B1) were provided by Sino Biological. .. The recombinant CR3022 therapeutic antibody was purchased from Creative Biolabs (MRO-1214LC).

    Article Title: Novel ACE2-Independent Carbohydrate-Binding of SARS-CoV-2 Spike Protein to Host Lectins and Lung Microbiota
    Article Snippet: For these three molecules, we used a concentration of 40μg/mL to coat the ELISA wells. .. Siglecs ELISA a solution of 50 µL of SARS-CoV-2 spike protein (2019-nCoV Spike RBD-His Recombinant Protein, Cat: 40592-V08H, expressed in HEK293 cells, purchased from Sinobiological) at 5 µg/mL, in PBS (10mM, pH=7.4), were used to coat the Nunc MaxiSorp plate 2h at 37°C. .. After discarding and washing (2×150µL) with Hanks’ Balanced Salt solution (Gibco™ HBSS) the wells were blocked with 200 µL of carbo-free blocking solution (Vector Laboratories, catalog No.NC9977573) at 37°C for 30 min.

    Concentration Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: 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). .. Recombinant His-tagged SARS-CoV-2 RBD protein (40592-V08H; Sino Biological) at a concentration of 10 µg/ml was loaded on Anti-Penta-HIS (HIS1K) Biosensors (FortéBio), followed by a washing step with assay buffer to block the unoccupied sensor surface. .. The association and dissociation profiles of imatinib (Sigma Aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µM to 0.78 µM).

    Blocking Assay:

    Article Title: Bcr-Abl tyrosine kinase inhibitor imatinib as a potential drug for COVID-19
    Article Snippet: 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). .. Recombinant His-tagged SARS-CoV-2 RBD protein (40592-V08H; Sino Biological) at a concentration of 10 µg/ml was loaded on Anti-Penta-HIS (HIS1K) Biosensors (FortéBio), followed by a washing step with assay buffer to block the unoccupied sensor surface. .. The association and dissociation profiles of imatinib (Sigma Aldrich) were measured at various concentrations (four-point serial dilutions from 6.25 µM to 0.78 µM).

    Microarray:

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform
    Article Snippet: .. Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.). .. On the same biochip, 7.5 μg/mL human IgG and 50 μg/mL BSA-biotin (Thermo Fisher Scientific) were also printed to serve as a printing control and “intra-well signal normalizer”, respectively.

    Binding Assay:

    Article Title: High-Accuracy Multiplexed SARS-CoV-2 Antibody Assay with Avidity and Saliva Capability on a Nano-Plasmonic Platform
    Article Snippet: .. Multiplexed SARS-CoV-2 microarray printing on pGOLD slidesEach pGOLD slide (Nirmidas Biotech Inc.) was printed with two SARS-CoV-2 antigens, namely the spike protein S1 subunit (S1) and S1 containing the receptor binding domain (RBD), using a GeSiM Nano-Plotter 2.1 at the following concentrations: 60 μg/mL for S1 (40591-V08H, Sino Biological Inc.) and 25 μg/mL for RBD (40592-V08H, Sino Biological Inc.). .. On the same biochip, 7.5 μg/mL human IgG and 50 μg/mL BSA-biotin (Thermo Fisher Scientific) were also printed to serve as a printing control and “intra-well signal normalizer”, respectively.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity
    Article Snippet: .. The stock S-RBD (2.5 μg/mL; 93.28 nM) was used to coat ELISA plates (Sino Biological 40592-V08H). .. The stock N-protein (1.25 μg/mL; 26.55 nM) was used to coat ELISA plates (Sino Biological 40588-V08B).

    Article Title: Novel ACE2-Independent Carbohydrate-Binding of SARS-CoV-2 Spike Protein to Host Lectins and Lung Microbiota
    Article Snippet: For these three molecules, we used a concentration of 40μg/mL to coat the ELISA wells. .. Siglecs ELISA a solution of 50 µL of SARS-CoV-2 spike protein (2019-nCoV Spike RBD-His Recombinant Protein, Cat: 40592-V08H, expressed in HEK293 cells, purchased from Sinobiological) at 5 µg/mL, in PBS (10mM, pH=7.4), were used to coat the Nunc MaxiSorp plate 2h at 37°C. .. After discarding and washing (2×150µL) with Hanks’ Balanced Salt solution (Gibco™ HBSS) the wells were blocked with 200 µL of carbo-free blocking solution (Vector Laboratories, catalog No.NC9977573) at 37°C for 30 min.

    Mouse Assay:

    Article Title: A Lymph Node Targeted Amphiphile Vaccine Induces Potent Cellular and Humoral Immunity to SARS-CoV-2
    Article Snippet: .. Mice were injected with 1 nmol CpG (soluble CpG), 1 nmol lipid-conjugated CpG (AMP-CpG), or 100 μg Alum admixed with phosphate-buffered saline (PBS) only (adjuvant controls), or 1-10 μg of Spike RBD protein (Sino Biological, Cat: 40592-V08H or GenScript, Cat: Z03483). ..

    Injection:

    Article Title: A Lymph Node Targeted Amphiphile Vaccine Induces Potent Cellular and Humoral Immunity to SARS-CoV-2
    Article Snippet: .. Mice were injected with 1 nmol CpG (soluble CpG), 1 nmol lipid-conjugated CpG (AMP-CpG), or 100 μg Alum admixed with phosphate-buffered saline (PBS) only (adjuvant controls), or 1-10 μg of Spike RBD protein (Sino Biological, Cat: 40592-V08H or GenScript, Cat: Z03483). ..

    Produced:

    Article Title: Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library
    Article Snippet: .. A Phage Library Display Panning Human synthetic Fab phage display libraries produced in-house (KFab-I and KFab-II, respectively built on human VH3/Vk1 and human VH1/Vk1 germline-based scaffolds, with randomized complementarity-determining regions) were used for the selection of specific binders against a SARS-CoV-2 spike protein (SARS-2 RBD) (Sino Biological, Cat. 40592-V08H, Beijing, China). .. The SARS-2 RBD was coupled to beads following the protocol for dynabeads (Thermofisher Scientific, Cat. 14301, Waltham, MA, USA).

    Selection:

    Article Title: Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library
    Article Snippet: .. A Phage Library Display Panning Human synthetic Fab phage display libraries produced in-house (KFab-I and KFab-II, respectively built on human VH3/Vk1 and human VH1/Vk1 germline-based scaffolds, with randomized complementarity-determining regions) were used for the selection of specific binders against a SARS-CoV-2 spike protein (SARS-2 RBD) (Sino Biological, Cat. 40592-V08H, Beijing, China). .. The SARS-2 RBD was coupled to beads following the protocol for dynabeads (Thermofisher Scientific, Cat. 14301, Waltham, MA, USA).

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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
    https://www.bioz.com/result/sars cov 2 rbd/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    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

    Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.

    Journal: bioRxiv

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    doi: 10.1101/2020.06.15.153064

    Figure Lengend Snippet: Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Polymerase Chain Reaction

    AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.

    Journal: bioRxiv

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    doi: 10.1101/2020.06.15.153064

    Figure Lengend Snippet: AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Chromatin Immunoprecipitation, Negative Control, Binding Assay, Derivative Assay

    Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.

    Journal: bioRxiv

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    doi: 10.1101/2020.06.15.153064

    Figure Lengend Snippet: Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Enzyme-linked Immunosorbent Assay

    Representative AIR array images (100 ms exposures) of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Figure 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.

    Journal: bioRxiv

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    doi: 10.1101/2020.06.15.153064

    Figure Lengend Snippet: Representative AIR array images (100 ms exposures) of (A) 5% FBS; (B) 10% PNHS; (C) a negative single-donor sample, and (D) one convalescent serum sample. Strong responses to SARS-CoV-2 antigens are readily observed in (D), but not in (A), (B), or (C). In each case, samples were diluted 1:20 in Adarza diluent, and incubated with the arrays overnight at 4 °C. See Figure 1 for key to the array. All arrays in this figure were imaged at an exposure of 100 ms.

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Incubation

    Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.

    Journal: bioRxiv

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    doi: 10.1101/2020.06.15.153064

    Figure Lengend Snippet: Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.

    Article Snippet: Calculated limits of detection for these data were 43.3 ng/mL (SARS-CoV-2 S1 + S2 ECD), 40.7 ng/mL (SARS-CoV-2 S1), and 25.1 ng/mL (SARS-CoV-2 RBD).

    Techniques: Titration, Concentration Assay, Standard Deviation

    Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Journal: Analytical Chemistry

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    doi: 10.1021/acs.analchem.0c01394

    Figure Lengend Snippet: Aptamers selection against the RBD of the SARS-CoV-2 spike glycoprotein.

    Article Snippet: SELEX Procedures We performed the aptamer selection procedure for SARS-CoV-2 RBD in a manner similar to our previous work.

    Techniques: Selection

    Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Journal: Analytical Chemistry

    Article Title: Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

    doi: 10.1021/acs.analchem.0c01394

    Figure Lengend Snippet: Results of docking and molecular dynamics simulations. (A) The overall structures of the CoV2-RBD-1C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue) (E) and the CoV2-RBD-4C aptamer (cyan) and the SARS-CoV-2 S protein complex (blue). (B) Detailed analysis of the interface between CoV2-RBD-1C and RBD (F) and the interface between CoV2-RBD-4C and RBD. Hydrogen bonds are shown by red, dashed lines. The amino acids of SARS-CoV-2-RBD targeted by aptamers are shown in blue, and the amino acids of SARS-CoV-2-RBD targeted by ACE2 are shown in red. (C) and (G) Flow cytometry results show that mutants with binding sites deleted exhibited significantly lower binding performance against RBD-Ni-beads compared to (C) CoV2-RBD-1C or (G) CoV2-RBD-4C aptamers. The lines represent the bases that were deleted. (D) and (H) The normalized binding efficiency of aptamers against RBD, under control or competition by ACE2: (D) for CoV2-RBD-1C and (H) CoV2-RBD-4C aptamers.

    Article Snippet: SELEX Procedures We performed the aptamer selection procedure for SARS-CoV-2 RBD in a manner similar to our previous work.

    Techniques: Flow Cytometry, Binding Assay

    S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: S309-CAR-NK cells are superior to CR3022-CAR-NK cells. ( a ) Diagram of S309 and CR3022 neutralizing antibodies binding to different epitopes of the SARS-CoV-2 S protein. Both open and closed conformation states of SARS-CoV-2 S protein are shown. S309 binding site is indicated in magenta and CR3022 binding site is indicated in yellow. ( b ) Quantitative data of CD107a surface expression of both S309-CAR-NK-92MI and CR3022-CAR-NK-92MI. Both transient 293T-hACE2-RBD and stable A549-Spike cell lines were used as target cells. Error bars represent SEM from at least two independent experiments. ( c ) Comparison of killing activity of S309-CAR and CR3022-CAR using the 4-hour Cr 51 release assay. Effector cells were cocultured with Cr 51 -labeled target cells at 37°C for 4 hours. The assay was repeated for at least two times per target cell line. ( d ) Expanded S309-CAR-NK primary has increased killing activity against A549-Spike cells than primary CR3022-CAR-NK primary . Effector cells were blocked with anti-CD16 and anti-NKG2D prior to coculturing with A549-Spike target cells for 4 hours at 37°C. Data were pooled from three independent experiments. Unpaired Student’s t test was employed for all panels. ns p > 0.05, * p

    Article Snippet: A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

    Techniques: Binding Assay, Expressing, Activity Assay, Release Assay, Labeling

    Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Journal: bioRxiv

    Article Title: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells

    doi: 10.1101/2021.01.14.426742

    Figure Lengend Snippet: Increased CD107a surface expression and killing activity of S309-CAR-NK-92MI cells against 293T-hACE2-RBD and A549-Spike target cells. (a) Generation of transient 293T-hACE2-RBD and stable A549-Spike cell lines. 293T-hACE2 cells were transfected with RBD-containing plasmid for 48 hours. Transfected 293T-hACE2-RBD cells were then harvested. For the generation of A549-Spike, 293T cells were transfected with the retrovirus transfection system for 48 hours. The spike retrovirus was filtered and transduced into A549 cells for an additional 48-72 hours. ( b ) Representative dot plots showing the expressions of RBD or Spike in 293T-hACE2 or A549 cells, respectively. 293T-hACE2-RBD and A549-Spike cells were stained with anti-RBD and the expressions were confirmed by flow cytometry. The stable A549-Spike cell line was then sorted to achieve high levels of spike expression. ( c ) Quantitative data of CD107a surface expression assay of S309-CAR-NK against 293T-hACE2-RBD or A549-Spike cell lines. Briefly, S309-CAR-NK-92MI cells were cocultured with either 293T-hACE2-RBD cells, A549-Spike cells, stimulated with PMA/Ionomycin, or incubated alone for 2 hours at 37°C. Cells were then harvested and stained for CAR F(ab)2 domain [IgG (H+L)] and CD107a. Data represent mean ± SEM from two experiments. ( d ) 4-hour standard Cr 51 release assay of S309-CAR-NK-92MI and parental NK-92MI cells against various target cell lines. 293T-hACE2-RBD, A549-Spike, and HepG2 cell lines were used as target cells for S309-CAR-NK and NK-92MI. Experimental groups were performed in triplicates. Error bars represent mean ± SEM from at least two independent experiments. Unpaired Student’s t test was used for both panels ( c ) and ( d ). ns p > 0.05, * p

    Article Snippet: A549-Spike cells were cultured for a few days prior to sorting using anti-RBD.

    Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Incubation, Release Assay