The recombinant S-RBD (Sino Biological, Beijing, China) and hACE2 protein (Sino Biological, Beijing, China) were used for surface plasmon resonance (SPR) analysis using a Biacore 8K instrument (Biacore, Uppsala, Sweden) as previously described (Chu et al., 2018a (link); Chu et al., 2018b (link)). Each target was immobilized onto flow cells in a CM5 sensor chip (GE Healthcare) via the amine-coupling method. Briefly, S-RBD was diluted in 10 mM pH 5.5 acetate to 20 µg/ml, while hACE2 was diluted in 10 mM pH 4.5 acetate to 20 µg/ml. Then the protein solutions were injected individually on the carboxyl modified sensor surface to form amine bonds. Both S-RBD and hACE2 immobilized levels were about 10,000 RU. Binding analyses were carried out at 25 °C and a flow rate of 30 μl min−1. The retrieved drugs in a running buffer (1×PBS, 0.05% Tween 20 and 5% dimethyl sulfoxide, pH 7.4) were run over each target at gradient concentrations as indicated. An empty flow cell, without any immobilized protein, was used as a deducted reference. The binding curves were analyzed using a kinetic binding model supplied with Biacore Evaluation Software (GE Healthcare).
Hace2 protein
Manufactured by Sino Biological
Sourced in China
HACE2 protein is a recombinant protein produced by Sino Biological. It is a member of the HECT-type E3 ubiquitin ligase family and functions as an enzyme that catalyzes the addition of ubiquitin to target proteins, marking them for degradation.
Automatically generated - may contain errors
Lab products found in correlation
Opteia assay diluent, by BD (2 mentions)
Biacore evaluation software, by GE Healthcare (1 mentions)
Cm5 sensor chip, by GE Healthcare (1 mentions)
Biacore 8k instrument, by Cytiva (1 mentions)
Camostat, by Bio-Techne (1 mentions)
Decanoyl rvkr cmk, by R&D Systems (1 mentions)
Hek293 cells, by Sino Biological (1 mentions)
Colorimetric microplate reader, by Bio-Rad (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Beyotime (1 mentions)
Microtiter plate, by Corning (1 mentions)
8 protocols using hace2 protein
1
Kinetic Characterization of SARS-CoV-2 S-RBD Binding to hACE2
2
SARS-CoV-2 RBD Binding Inhibition Assay
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The ability of antibodies to inhibit the binding of the SARS-CoV-2 RBD to hACE2 was investigated by ELISA. The 96-well ELISA plates were coated with 200 ng of hACE2 protein (Sino Biological, 10108-H08H) per well overnight at 4°C and washed with PBST and blocked for 1 h with blocking buffer as above. Meanwhile, 5-fold serial dilutions of MAbs or bispecific antibodies or a cocktail were incubated with 4 ng/mL SARS-CoV-2 RBD with mouse IgG Fc tag protein (Sino Biological, 40592-V05H) for 1 h at 25°C. Then, the mixtures were added to ELISA plates and incubated for 1 h at 37°C. After further washing, bound SARS-CoV-2 RBD protein was detected with anti-mouse Fc HRP antibody (Abcam) diluted 1:10000 in blocking solution, followed by washing with PBST. The ELISA plates were reacted with TMB substrate at 25°C for 5 min and then stopped by 0.2 M H2SO4 stop buffer and determined at OD 450 nm. The IC50 was determined by using 4-parameter logistic regression.
3
SARS-CoV-2 Infection Inhibition Assay
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The reduction of luciferase gene expression was detected to evaluate the infection inhibition effect of hACE2 protein (expressed in HEK293 cells, Sino Biological), protease inhibitor Decanoyl-RVKR-CMK (Furin inhibitor, R&D Systems), protease inhibitor Camostat (TMPRSS2 inhibitor, Tocris Bioscience), protease inhibitor E64D (Cathepsin L inhibitor, APExBIO), monoclonal antibody, and sera. As described previously19 (link),20 (link), the tested samples were diluted (starting with 30 times dilution and three times gradient, a total of eight gradients), after which the virus solution was added. On each 96-well plate, eight virus control wells and eight cell control wells were arranged. In the virus control wells, no test sample but only virus solution was added; in the cell control wells, no virus solution but only a complete culture medium was added. The 96-well plates were incubated at 37 °C for 1 h, after which trypsinized Huh7 cells (2 × 104/100 μL) were added to each well. After incubation for 24 h in an atmosphere comprising 5% CO2 at 37 °C, luminescence was measured as described above. The EC50 value of each sample was calculated using the Reed–Muench method.
4
SARS-CoV-2 Spike Protein Binding Assay
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The SARS‐COV‐2 extracellular spike protein (S1+S2 ECD), the S1 domain of S, the hACE2 protein were purchased from Sino Biological (Beijing, China).
5
Biotinylated hACE2 Binding Assay
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Biotinylated hACE2 was synthesized by incubating a 1:1 molar ratio of succinyl propionyl biotin (Dojin Chemical, Kumamoto, Japan, 346-06351) and hACE2 protein (SinoBiologicals, Wayne, NJ, USA. 10108H08H) on ice, overnight and at room temperature, for 1 h. A 50 μL aliquot containing 100 ng RBD protein was added to each well of a 96-well microtiter plate (NUNC, 44-2404-21), followed by overnight incubation at 4 °C. The plates were blocked by incubation with 3% BSA in PBS, followed by incubation with an appropriate dilution of biotinylated hACE2 on ice for 30 min. After washing with PBST, HRP-conjugated streptavidin was added to each well, and the plate was incubated for 30 min. The plate was washed four times with PBST, followed by the addition of the TMB substrate solution. The enzymatic reaction was stopped by the addition of 0.2 N H2SO4, and the absorbance of each well at 450 nm was measured with a colorimetric microplate reader (BioRad).
6
SARS-CoV-2 RBD Protein Binding Assay
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Two hundred nanograms of hACE2 protein (Sino Biological, 10108-H05H) in 50 μl PBS per well was coated on ELISA plates overnight at 4°C. Then, the ELISA plates were blocked for 1 h with blocking buffer (5% FBS plus 0.05% Tween 20); meanwhile, threefold serial diluted mAbs or twofold diluted patient sera were incubated with 0.2 μg/ml SARS-CoV-2 RBD protein for 1 h. Then, the incubated mixtures were added to ELISA plates and allowed to develop for 1 h, followed by PBST washing and anti-His HRP antibody (Sino Biological, 105327-MM02T-H) incubating for 45 min. Next, the ELISA plates were washed with PBST and added with TMB (Beyotime). After 5 min, the ELISA plates were stopped and determined at 450 nm. The half maximal inhibitory concentration (IC50) was determined by using four-parameter logistic regression.
7
SARS-CoV-2 Neutralizing Antibody Assay
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The surrogate virus based neutralization test (sVNT) was used for measuring neutralizing antibody (sNAb) to SARS-CoV-2 as previously described [25 (link)]. Briefly, microtiter plates were coated overnight with 2 μg/ml hACE2 protein (Sino Biological, China) at 4°C, followed by blocking with OptEIA assay diluent (BD). The HRP–RBD conjugate (3 ng) was pre-incubated with 100 μl of diluted serum samples for 1 h at 37°C, then added into hACE2 pre-coated plate for 1 h at room temperature. Plates were washed five times by PBST. A colorimetric signal was developed with TMB, and equal volume of stop solution was added to terminate the reaction. The absorbance reading was performed at 450 nm and 570 nm. Inhibition rate (%) = (1 − sample optical density value/negative control optical density value) × 100.
8
Surrogate Virus Neutralization Assay
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The surrogate virus based neutralization test (sVNT) was used for measuring neutralizing antibody (NAb) to SARS-CoV-2 as previously described (28). Briefly, microtiter plates (Corning) were coated overnight with 2μg/ml hACE2 protein (Sino Biological, Beijing, China) at 4 °C, followed by blocking with OptEIA assay diluent (BD). The HRP-RBD conjugate (3 ng) was pre-incubated with 100 μl of diluted serum samples for 1 h at 37 °C, then added into hACE2 pre-coated plate for 1 h at room temperature. Plates were washed five times by PBST. A colorimetric signal was developed with TMB, and equal volume of stop solution was added to terminate the reaction. The absorbance reading was performed at 450 nm and 570 nm. Inhibition rate (%) = (1 -sample optical density value/negative control optical density value) × 100.
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