| Assay Method Information | |
| | Activity of Compounds as TLR7 and TLR8 Agonists as Measured in Reporter Cell Assays |
| Description: | The engagement of TLRs by cognate ligands triggers downstream signaling cascades, leading to the activation of NF-κB and other transcription factors which initiate various immunomodulatory effects. The human embryonic kidney cell line, HEK293, is essentially non-responsive to TLR agonists, but ectopic expression of TLRs in these cells allows cognate agonists to activate endogenous NF-κB. Accordingly, the HEK293-TLR-NF-κB inducible reporter system is used to assay TLR agonists. HEK293 cell lines stably expressing human TLR7 or TLR8 together with an NF-κB-driven-secreted alkaline phosphatase (SEAP) reporter were invented at InvivoGen (San Diego, Calif., USA) and used to assess compounds of the present invention for TLR7 and TLR8 agonist activities. Cells were seeded at 2-5×104 cells/well in 96 well plates (200 l/well) and treated with various concentrations of compound (10 μl) for 15-24 hours. SEAP activity was determined by measuring OD at 650 nm; the media used to culture the cell lines contains the reagents required for SEAP detection. The EC50 values in Table 2 are calculated from fitting the dose response of measured SEAP activity for each compound to the following equation: Y=ymax*cnh/(EC50 nh+cnh)+blank where Y is the experimentally measured OD650 at concentration c of test article, blank is the observed OD650 in the absence of TLR7 agonist, ymax is the difference between the measured OD650 in the presence of either 28.5 μM resiquimod or 1.675 μM CL307 and the blank and values of EC50 and nh are determined by nonlinear least squares analysis. |
| Affinity data for this assay | |
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