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
Index: Curr. Chem. Genomics 5 , 21-9, (2011)
Full Text: HTML
Abstract
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.
Related Compounds
Related Articles:
Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair.
2012-02-10
[Mol. Cell. 45 , 371-383, (2012)]
Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance.
2006-11-01
[Nat. Chem. Biol. 2 , 608-617, (2006)]
2010-01-01
[PLoS Biol. 8 , (2010)]
2009-05-05
[PLoS Biol. 7 , e1000111, (2009)]