Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: Viruses

Article Title: Design of Three Residues Peptides against SARS-CoV-2 Infection

doi: 10.3390/v14102103

Figure Lengend Snippet: BLI interaction analysis. RBD (0.2 µM) ( A , B ) either alone in solution or mixed with VFI ( A ) and TLH ( B ) peptides, tested at different concentrations (40 µM, blue lines; 80 µM, green lines and 160 μM, orange lines). BLI interaction was performed at 25 °C in PBST (10 mM phosphate, 150 mM NaCl, 0.05% Tween 20, pH 7.4). The corresponding plots ( C , D ) of steady-state binding from the end of the association phases (nm), after the subtraction of RBD signal, against analyte concentration were used to calculate the steady-state affinity by nonlinear regression analysis using GraphPad 5 software.

Article Snippet: An Octet ® Red 96 system ® (ForteBio, Fremont, CA, USA) was used to detect interactions between the His-tagged Spike RBD protein (Elabscience Biotechnology, Biomedical Park, Wuhan, China; Cat. No.: PKSH032068) and peptides using Octet His2 biosensors according to the standard instructions with minor modifications.

Techniques: Binding Assay, Concentration Assay, Software

Journal: medRxiv

Article Title: Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current

doi: 10.1101/2020.11.24.20237628

Figure Lengend Snippet: Schematic illustration of SARS-CoV-2 spike protein and ACE2 receptor binding. (a) SARS-CoV-2 binding to the ACE2 receptor on the host cell surface. (b) Binding of ACE2 and spike protein along with illustration of the spike protein subunits, S1 and S2. (c) Schematic showing the distribution of the spike protein in solution. (d) Schematic showing the distribution of ACE2 in suspension. (e) Distribution of ACE2 and S protein after binding in solution.

Article Snippet: The human ACE2 recombinant protein (ProSci Cat No. 10-114) was resuspended in sterile deionized water at a stock concentration of 500 ng/µl as per the manufacturers instruction and used for the analysis.

Techniques: Binding Assay

Journal: medRxiv

Article Title: Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current

doi: 10.1101/2020.11.24.20237628

Figure Lengend Snippet: Optical detection of binding interactions between ACE2 and other proteins. (a) Measured light intensities over time for individual assessment of ACE2, S1X, S1Y, and BSA. (b) The measured mixed light intensities versus time for ACE2 mixed with either S1X, or S1Y, or BSA. (c) The measured ACE2-S1X interaction profile for an extended time period.

Article Snippet: The human ACE2 recombinant protein (ProSci Cat No. 10-114) was resuspended in sterile deionized water at a stock concentration of 500 ng/µl as per the manufacturers instruction and used for the analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

doi: 10.3390/ijms23052643

Figure Lengend Snippet: Inhibitory effects of different polyphenols on the interaction between SARS-CoV-2 spike protein receptor binding domain (RBD (N501Y)) and human angiotensin-converting enzyme 2 (ACE2). ( A ) 10 µM of pelargonidin-3-O-glucoside (Pel-3-O-G), malvidin-3-O-glucoside (Mal-3-O-G), cyanidin-3-O-glucoside (Cya-3-O-G), peonidin, tannic acid (TA), 1,3,6-tri-O-galloyl-β-D-glucose (TGG), and corilagin were tested to evaluate their ability to inhibit the binding of immobilized spike protein (0.5 µg/mL) to human, biotin-labeled ACE2 (0.25 µg/mL) by using an enzyme-linked immunosorbent assay (ELISA). Dose effect inhibition of 0.1, 1, and 5 µM ( B ) TA, ( C ) TGG, and ( D ) corilagin. The absorbance of ACE2 (0.25 µg/mL) at 450 nm was set to 100%. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test with * p < 0.05, ** p < 0.01, *** p < 0.001 compared to ACE2 (0.25 µg/mL).

Article Snippet: Biotinylated recombinant human ACE2 with purity >95% and camostat mesylate compound were both purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Labeling, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: bioRxiv

Article Title: Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation

doi: 10.1101/2021.01.13.426436

Figure Lengend Snippet: A cellular immune response and neutralizing antibodies. A) IFN-γ and IL-4 ELISpot responses in splenocytes from rats vaccinated with 240 μg pVAX1-SARS-CoV2-co, with or without re-stimulation with recombinant S1+S2. The p-value calculated using Welch’s t-test between IFN-γ secreting cell number in the antigen-stimulated group and the un-stimulated group was 0.0059 (n=6). B) ACE2 (and S1+S2) binding inhibition assay using 2-to 12-week immunized rat serum normalized to pre-serum, at reciprocal 10-to 31250-fold dilutions. C) Neutralization titers (ID75) of 2-to 12-week immunized rat serum indicated as the dilution of serum required for 75% inhibition of ACE2-S1+S2 binding as shown in (B). D) Inhibition rate against the pseudovirus. The immunized rat serum at 8 weeks was used at reciprocal 20-to 12500-fold dilutions. E) Neutralization activity (ID75; 75% inhibition). The values indicate the inhibitory dose of the serum which shows 75% inhibition rate against pseudovirus binding as shown in (D).

Article Snippet: To analyze the binding inhibition of S1+S2 and ACE2 by neutralizing antibodies in the immunized rat and mouse serum, 96-well plates were coated with human ACE2 recombinant protein (1μg/ml, mFc tag; #83986, Cell Signaling Technology), and then blocked using PBS containing 5% skim milk for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Recombinant, Binding Assay, Inhibition, Neutralization, Activity Assay

Journal: bioRxiv

Article Title: Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation

doi: 10.1101/2021.01.13.426436

Figure Lengend Snippet: Virus challenge on pVAX1-SARS-CoV2-co immunized mice A) Pre-viral challenge of mice immunized with 160 μg of pVAX1-SARS-CoV2-co plasmid intradermally (ID; n = 8) or no-vaccinated (NV: n=10) two times at 2-week intervals. Antibody titer (half maximum) for recombinant S1+S2 in the blood serum 16 weeks post initial vaccination, assessed using ELISA. P <0.001 Student’s t -test. B) Antibody titer (half maximum) for recombinant RBD in the blood serum in ID and NV mice. P =0.018 Student’s t -test. C) Neutralization titer (ID75) for recombinant ACE2-S1+S2 binding inhibition in the blood serum of ID mice. D) ID and NV (n=10) mice were intranasally infected with mouse-adapted SARS-CoV-2 virus. 50% tissue culture infective doses (TCID50) values in the lung tissues of each animal are shown. P =0.016 Student’s t -test. All individual values are shown in .

Article Snippet: To analyze the binding inhibition of S1+S2 and ACE2 by neutralizing antibodies in the immunized rat and mouse serum, 96-well plates were coated with human ACE2 recombinant protein (1μg/ml, mFc tag; #83986, Cell Signaling Technology), and then blocked using PBS containing 5% skim milk for 2 h at room temperature.

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Neutralization, Binding Assay, Inhibition, Infection

Journal: bioRxiv

Article Title: Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation

doi: 10.1101/2021.01.13.426436

Figure Lengend Snippet: Individual values of pre-viral challenge antibody titers of ID and NV mice for recombinant S1+S2 (A), RBD (B) and neutralization titer (inhibitory dose at 50% neutralization; ID50) of ACE2-S1+S2 binding inhibition (C), as well as TCID50 values in the mouse lung tissues after virus challenge as described in

Article Snippet: To analyze the binding inhibition of S1+S2 and ACE2 by neutralizing antibodies in the immunized rat and mouse serum, 96-well plates were coated with human ACE2 recombinant protein (1μg/ml, mFc tag; #83986, Cell Signaling Technology), and then blocked using PBS containing 5% skim milk for 2 h at room temperature.

Techniques: Recombinant, Neutralization, Binding Assay, Inhibition, Virus

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: ACE2 substrate/assay buffer solution a

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques:

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Components of ACE2 activity standard curve solution (per well) a

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Activity Assay

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Standard curves for ACE2 activity assay. The assay was performed for 16 h incubation. Graph shows the concentrations of recombinant ACE2 (rACE2) versus the ACE2 activity (RFU). ( a ) Standard curves generated for human rACE2. The ACE2 inhibitors MLN-4760 (10 −6 M) or DX600 (10 −6 M) were used. The relationship is highly linear for rACE2 concentrations between 0 and 50 ng/ml ( R 2 = 0.9998, p < 0.001 for MLN-4760, R 2 = 0.9999, p < 0.001 for DX600). ( b ) Standard curve generated for mouse rACE2. The ACE2 inhibitor MLN-4760 (10 −6 M) was used. A highly linear relationship exists for rACE2 concentrations between 0 and 50 ng/ml ( R 2 = 0.9976, p < 0.001)

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Activity Assay, Incubation, Recombinant, Generated

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Intra- and inter-assay coefficients of variability (CV)

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Inter Assay, Activity Assay, Intra Assay, Clinical Proteomics

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Effect of ACE2 inhibitors MLN-4760 (10 −6 M), DX600 (10 −6 M), and ACE inhibitor captopril (10 −5 M) on ACE2 enzyme activity. The assay was performed for 16 h incubation. Graph depicts the concentrations of recombinant ACE2 versus the Relative Fluorescence Units (RFU). A background RFU value (67.5 RFU for a , 62.0 RFU for b ) of the substrate blank control was subtracted from the readings for each well. ( a ) Effect of MLN-4760, DX600, and captopril on human rACE2 activity. ( b ) Effect of MLN-4760, DX600, and captopril on mouse rACE2 activity

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Activity Assay, Incubation, Recombinant, Fluorescence, Control

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Effect of substrate concentrations on ACE2 activity. ( a ) The assay was performed with different ACE2 substrate concentrations (11.25, 5.63, 2.81, and 1.41 μM) for 16 h incubation using mouse rACE2 concentrations between 0 and 200 ng/ml. Graph depicts the concentrations of mouse rACE2 versus the activity (RFU) at different substrate concentrations. ( b ) Standard curves generated for mouse rACE2 with 11.25 or 5.63 μM ACE2 substrate concentrations. A highly linear relationship between mouse rACE2 and the RFU exists for both substrate concentrations ( R 2 = 0.9994, p < 0.001 for 11.25 μM; R 2 = 0.9903, p < 0.001 for 5.63 μM)

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Activity Assay, Incubation, Generated

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Time course of ACE2 activity assay. ACE2 activity was measured for 2, 6, 16, and 24 h incubation, with mouse rACE2 concentrations ranging from 0 to 200 ng/ml. Graph depicts the concentrations of mouse rACE2 versus the ACE2 activity (RFU) at different time points. ( a ) Standard curve generated for mouse rACE2 for 2 h incubation. A highly linear relationship exists for rACE2 concentrations from 0 to 200 ng/ml ( R 2 = 0.9981, p < 0.001). The area marked with dashed lines is shown in b . ( b ) Standard curve generated for mouse rACE2 for 16 h incubation. The relationship is highly linear for rACE2 concentrations between 0 and 50 ng/ml

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Activity Assay, Incubation, Generated

Journal: Hypertension

Article Title: Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2)

doi: 10.1007/978-1-4939-6625-7_8

Figure Lengend Snippet: Effect of potential modulators on ACE2 activity assay. The assay was performed for 16 h incubation. ( a ) Graph shows ACE2 activity in defined medium (not incubated with mouse PT cells), and culture medium collected from primary cultures of mouse PT cells after 72 h incubation, in the presence of normal (7.8 mM) or high concentrations of d -glucose (HG, 25 mM). No ACE2 activity is detected in the defined medium. Results are means ± SEM; p > 0.05, culture medium vs culture medium + HG; n = 3. ( b ) Graph depicts the effect of addition of various concentrations of creatinine on ACE2 activity (RFU) at different mouse rACE2 concentrations. Results are means ± SEM; p > 0.05 for all graphs; n = 3. ( c ) Graph depicts the effect of addition of various concentrations of urea on ACE2 activity (RFU) at different mouse rACE2 concentrations. Results are means ± SEM; p > 0.05 for all graphs; n = 3. ( d ) Graph depicts the effect of addition of exogenous albumin on ACE2 activity (RFU) at different mouse rACE2 concentrations. Albumin at the indicated concentrations was added to urine samples (1 μl) from mice with gene deletion of ACE2. Results are means ± SEM; p > 0.05 for all graphs; n = 3

Article Snippet: ACE2 Standard: Recombinant human ACE2 (human rACE2, R&D Systems Inc., Minneapolis, MN, USA, Catalog Number: 933-ZN) and recombinant mouse ACE2 (mouse rACE2, R&D Systems Inc., Catalog Number: 3437-ZN).

Techniques: Activity Assay, Incubation