| Assay Method Information | |
| | Cytochrome P450 Isoenzyme Inhibitory Activity Test |
| Description: | The inhibitory activities of test compound against different isoforms of human cytochrome P450 isoenzymes were determined. Experimental Operation. The test compound, standard inhibitor (100×final concentration) and mixed substrate working solution were prepared; the microsome frozen in −80° C. refrigerator was taken out and thawed. 2 μL of the compound to be tested and standard inhibitor solution were added to the corresponding wells, and at the same time, 2 μL of the corresponding solvent was added to the non-inhibitor control wells (NIC) and the blank control wells; secondly, 20 μL of mixed substrate solution was added to the corresponding wells except the blank wells (20 μL of Pb was added to the blank wells); human liver microsome solution was prepared (the solution was put back in the refrigerator immediately after using and marking the date), and then 158 μL of human liver microsome solution was added to all wells; the sample plate was put in a 37° C. water bath for pre-incubation, and then a coenzyme factor (NADPH) solution was prepared; after 10 minutes, 20 μL of NADPH solution was added to all wells, the sample plate was shaken well, and incubated in a 37° C. water bath for 10 minutes; at the corresponding time point, 400 μL of cold acetonitrile solution (internal standard is 200 ng/mL tolbutamide and labetalol) was added to terminate the reaction; after the plates were evenly mixed, the mixture was centrifuged at 4000 rpm for 20 minutes to precipitate protein; 200 μL of supernatant was added into 100 μL of water, shaken well and detected by LC/MS/MS. |
| Affinity data for this assay | |
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