| Assay Method Information | |
| | AlphaLISA Assay |
| Description: | The ability of the compounds to block binding of IL-17A to its receptor, IL-17RA, was analysed in a competition assay using AlphaLISA technology (Perkin Elmer). The assay is a bead based AlphaLISA where the IL-17RA is captured on the acceptor bead via an Fc tag and IL-17A is captured on the streptavidin donor bead via a biotinylated anti-IL-17A antibody.Assay buffer was prepared by adding 0.05% Tween-20 (v/v) and 0.1% BSA to Phosphate Buffered Saline (PBS). The assay was carried out in 384-well white low volume plates (Corning 4512). 10 μL of a 7.5 nM stock of human recombinant IL-17A (R&D Systems 7955-IL/CF) diluted in assay buffer was dispensed into the assay plate and compounds or DMSO vehicle control were added in a volume of 75 nL using a D300 dispenser (Hewlett Packard). The compounds were pre-incubated with the IL-17A for 24 h at room temperature (or for 30 min, where indicated by * in Table A below) prior to addition of 5 μL of a 5 nM stock of human recombinant IL-17RA/Fc chimera (R&D Systems 177-IR-100) diluted in assay buffer. The IL-17A was incubated with the receptor for a further 90 minutes at room temperature before addition of 5 μL of a mixture of anti-human Fc IgG acceptor beads (75 μg/mL, Perkin Elmer AL103C) and anti-IL-17A biotin conjugated antibody (5 nM, Enzo Life Sciences, ENZ-ABS278-0100) in assay buffer. After a further 30 min incubation at room temperature, 5 μL of streptavidin donor beads (75 μg/mL, Perkin Elmer 6760002S) were added and the plate was incubated for 3 h in the dark. The luminescence signal was measured using an Enspire plate reader (Perkin Elmer) with excitation at 680 nm and emission at 615 nm. Data were analysed using GraphPad Prism and fitted to a 4-parameter logistic equation. |
| Affinity data for this assay | |
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