GlaxoSmithKline, Collegeville, PA, USA
5th HTRF Symposium, Avignon, France
384- or 1536-well HTRF® cell based assays for high throughput screening and XC50 compound profiling were developed to detect secreted IL-8, IL-1?, and TNF? cytokines. For IL-1? and TNF?, the HTRF® format was compared to the AlphaLISA format. The IL-1? assay was designed to detect cytokine production driven by the NLRP3 Pattern Recognition Receptor, and the IL-8 assays were designed to detect cytokine production that was driven through the NOD1 or NOD2 Pattern Recognition Receptors (PRRs). The TNF? assay was used as a specificity assay to help eliminate compounds not acting through the desired target. Cell lines were specifically chosen or engineered to produce a specific response through one of the receptors and PRR specific ligands were used to activate the pathways of interest. The NOD1 and NOD2 screening efforts allowed the identification of compounds selective for NOD1 and NOD2. The details of assay development and screening for these cellular HTRF® assays will be presented.