| Assay Method Information | |
| | Biochemical Assay |
| Description: | Compounds disclosed herein were tested for blocking of human IL-17A (Cat: C774, novoprotein) protein with its receptor human IL-17RA (Cat: C153, novoprotein) in an assay based on Homogeneous Time Resolved Fluorescence. 0.4 nM recombinant human IL-17A protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 10 uM, 2.7-fold serially diluted, 10 points) at room temperature for 3 hours in an assay buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl, 0.1% BSA, 0.2 mM DTT, 0.005% Tween 20. Then 0.3 nM recombinant human IL-17RA was added to plate and further incubated at room temperature for 1 hour. After that Mab Anti-6His Tb cryptate Gold (Cat: 61HI2TLB, Cisbio Bioassays) and MAb Anti Human IgG-XL665 (Cat: 61HFCXLB, Cisbio Bioassays) were added to plate and further incubated at room temperature for 1 hour. The HTRF signals (ex337 nm, em620 nm/665 nm) were read on BMG PHERAstar FSX instrument. The inhibition percentage of human IL-17A interaction with its receptor human IL-17RA in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 620 nm to that at 665 nm. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Dotmatics. |
| Affinity data for this assay | |
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