Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells
Kristin P Leister, Ruili Huang, Bonnie L Goodwin, Andrew Chen, Christopher P Austin, Menghang Xia*
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA
Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.
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* Address correspondence to this author at the National Institutes of Health, National Human Genome Research Institute, NIH Chemical Genomics Center, 9800 Medical Center Drive, Bethesda, MD 20892-3370, USA; Tel: 301-217-5718; Fax: 301-217-5736; E-mail: email@example.com