The quantity of RBD expressed in EBY200 surface was determined by quantitative ELISA, as described previously (23 (link)). In brief, 0.5 mg of dry power of S. cerevisiae pYD1-RBD/EBY200 cells was weighed in 1.5-ml microtubes and washed twice with PBS. Collected cell pellets were resuspended in 100 µl of a series of diluted Anti-Spike-RBD human IgG (Sanyou Bio, Shanghai, China) solutions [in PBS containing 1% (w/v) BSA] and incubated at room temperature for 1 h. Cell pellets were washed three times in PBS containing 0.05% Tween-20. After washing, cells were resuspended in 100 µl of PBS containing 0.1 µg of rabbit anti-human IgG-HRP antibody (Abcam, UK) and incubated at room temperature for 1 h. After washing in the same way, cells were developed with the addition of 100 µl of HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (YEASEN, Shanghai, China) at room temperature in darkness for 30 min and stopped reactions by addition of 100 μl of 2 mol/L H2SO4. Finally, absorbance of cell supernatant was measured at 450 nm using a microplate reader (Thermo Electron Corporation, USA).
Hrp substrate 3 3 5 5 tetramethylbenzidine tmb
Manufactured by Yeasen
Sourced in China
HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) and other immunoassays. It is commonly used to detect the presence and quantify the amount of target analytes in a sample when conjugated with a horseradish peroxidase (HRP) enzyme label.
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2 protocols using hrp substrate 3 3 5 5 tetramethylbenzidine tmb
1
Quantitative ELISA for RBD Expression
2
Quantifying RBD-ACE2 Binding Affinity
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To determine the affinity of RBD and its deglycosylated mutants to ACE2, 0.5 OD 600 nm of S. cerevisiae pYD1-RBD/EBY200 cell pellets was centrifuged in 1.5-ml microtubes and washed twice with PBS. Subsequently, cells were resuspended in 100 µl of a series of diluted biotinylated Human ACE2 protein (Acro Biosystems, Beijing, China) solutions in PBS containing 1% (w/v) BSA and incubated at room temperature for 1 h. Cell pellets were washed three times in PBS containing 0.05% Tween-20. After washing, cells were resuspended in 100 µl of PBS containing 0.1 µg of Streptavidin-HRP (Acro Biosystems, Beijing, China) and incubated at 37°C for 1 h to avoid light. After washing in the same way, cells were developed with the addition of 100 µl of HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (YEASEN, Shanghai, China) at room temperature in darkness for 30 min and reactions were stopped by the addition of 100 μl of 2 mol/L H2SO4. Finally, absorbance of cell supernatant was measured at 450 nm using a microplate reader.
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