| Assay Method Information | |
| | Enzyme (HIV Integrase) Inhibitory Activity Assay |
| Description: | The donor DNA was diluted with TE buffer to 10 nM, of which 50 μl was added to each well of streptavidin-coated microtiter plate (manufactured by Roche) and allowed to adsorb at 37° C. for 60 min. The DNA was then washed with phosphate buffer (Dulbecco PBS, Sanko Junyaku Co., Ltd.) containing 0.1% Tween 20 and phosphate buffer. Then, a reaction mixture (70 μl, see the following * for the composition), a test substance (10 μl) diluted with the reaction mixture and 100 g/ml integrase protein (10 μl) were added to each well and reacted at 37° C. for 60 min.Then, 50 nM target DNA (10 μl) was added, reacted at 37° C. for 10 min and washed with phosphate buffer containing 0.1% Tween 20 to stop the reaction.Then, 100 mU/ml peroxidase labeled anti-digoxigenin antibody solution (manufactured by Roche, 100 μl) was added, and the mixture was reacted at 37° C. for 60 min, followed by washing with phosphate buffer containing 0.1% Tween 20.A peroxidase color solution (manufactured by Bio Rad, 100 μl) was added and allowed to react at room temperature for 4 min. The color reaction was stopped by adding 1N sulfuric acid (100 μl). The absorbance at 450 nm was measured.The HIV integrase inhibitory activity (IC50) of the compound A of the present invention was calculated from the inhibition rate according to the following formula. The results are shown in Table 8.Inhibitionrate(%)=[1-(Object-Blank)/(Control-Blank)]×100 |
| Affinity data for this assay | |
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