Anti his hrp

Heparin (APP Pharmaceuticals) (50 ng) in 5 μL solution each was pipetted into each well of a high binding plate. A set of wells were set up without heparin. A solution (0.2 mL) of 90% saturated (3.7 M) ammonium sulfate was added to each well and incubated overnight to immobilize the HS. The next day, the wells were washed twice with 0.2 mL of PBS then blocked with 0.2 mL of PBS containing 0.1% Tween 20 (PBST) and 0.1% BSA for 1 h at room temperature. The wells were emptied and 45 μL of PBST/BSA with 0, 1, 3, 6, 10, 30, 60 or 100 nM of his/FLAG-tagged SARS-CoV-2 spike protein (GenScript, Z03481-100) was added to each well. The wells were incubated for 1 h at room temperature, washed thrice with 0.2 mL of PBST, and then incubated with 45 mL each of 0.1 μg/mL of THE anti-his-HRP (GenScript, A00612) in PBST/BSA for 1 h at room temperature. The wells were washed 5 times with 0.2 mL of PBST. TMB Turbo substrate was added to each well (0.1 mL), and the color was allowed to develop. The reaction was quenched by addition of 0.1 mL of 1 M sulfuric acid. The absorbance was measured by 450 nm.