Radioligand-based binding assays

The binding assays of Hood et al.⁶⁹ were used to assess the direct binding activity to COX-2 by measuring the competitive binding of the radiolabeled inhibitor [³H] celecoxib to the target enzyme. The murine monoclonal COX-2-specific antibody was coated onto 96-well Immulon 2HB microtiter plates (Thermo Scientific, Waltham, USA) and incubated overnight at 37 °C. The coated plates were washed with Dulbecco’s PBS (D-PBS) and blocked by 10% skim milk to avoid nonspecific binding. The recombinant COX-2 enzyme binding buffer was added to plates and incubated for 2 h at 37 °C and these antibody-captured enzyme-coated plates were washed with D-PBS. To measure the competitive binding activity with celecoxib, compounds at their IC₅₀ concentrations were incubated with [³H] celecoxib and allowed to compete for the binding to COX-2 for 2 h. After that, the incubation was halted by aspiration and washed twice with cold D-PBS. The 50 μl of 10% SDS was added into plates for 1 h at 37 °C. At last, the COX-2 bound radioligand was transferred into the liquid scintillation vial for quantitation using the liquid scintillation spectrometry.