Horseradish peroxidase hrp anti his tag antibody

Human ACE2 protein was immobilized onto a microtiter plate at 5 μg/ml (100 μl/well). Each S protein (prototype and V367F) was added as a ligand at different concentrations, ranging from 0.03 μg/ml to 10 μg/ml, and then incubated for 2 h at 37°C to allow receptor-ligand interaction. The ligand-receptor mixture was then washed three times. One hundred microliters of horseradish peroxidase (HRP) anti-His tag antibody (BioLegend, USA) (diluted 1:20,000) was added to each well and allowed to react for 1 h. After three washes, the signal was visualized using 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Sigma-Aldrich, USA), and a microtiter plate reader recorded the optical density at 450 nm (OD₄₅₀).

96 microtiter-plates were seeded with VERO-E6 or BEAS-2B cells at 20,000 cells/well. The cells were washed by PBS and fixed with 4% formaldehyde 1 day later. 3% H₂O₂ were added to quench endogenous peroxidase. Cells were then blocked with 1% BSA in PBS for 1 h at 37 °C. Serially concentrations (0–40 μg/mL) of COVID19-SF5 were added and incubated with the cells for 2 h at 37 °C. Horseradish peroxidase (HRP) anti-His Tag antibody (1:20,000, Biolegend, San Diego, USA) were added after washing. After another 3 times washing, the plates were developed with 3,3′,5,5′-tetramethylbiphenyldiamine (TMB) for 15 min. The reactions were stopped by the addition of 2 M H₂SO₄ stop solution. The absorbance was measured on a microplate reader at 450 nm.