Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

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.

Advances in high-throughput screening technology for toxicology

“…Two homogeneous cell-based TNF-α assays have been developed and optimised in a 1,536-well plate format using homogeneous time resolved fluorescence (HTRF) and AlphaLISA immunoassay technologies. Leister et al (2011) further validated these assays by conducting a LOPAC screen. The HTRF-based TNF-α assay requires labeling anti-TNF-α antibodies with HTRF donor or acceptor fluorophores.…”

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“…Thus, a causative role for TNFα is insinuated in many diseases including Crohn disease, inflammatory bowel disease, obesity, and type 2 diabetes. [13][14][15] TNFα mediates these responses by interaction with at least two distinct receptors ubiquitous to most cells, the high affinity TNF receptor 1 (TNFR1 or CD120a) and the low-affinity TNF receptor 2 (TNFR2 or CD120b). 16 Because of its role in disease, a multitude of therapeutic monoclonal antibodies to TNFα have been developed including infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), or the circulating receptor fusion protein etanercept (Enbrel).…”

“…The reaction produces a color change and absorbance of the color generated is read on a plate reader at a wavelength of 450 nm. 14,15 The absorbance of the colored product is directly proportional to the concentration of TNFα present in the samples. Another assay, although less convenient method to quantitate TNFα, is the radioisotope labeled immune assay.…”

“…Another assay, although less convenient method to quantitate TNFα, is the radioisotope labeled immune assay. 14 This assay is as cumbersome as the ELISA method, because it involves numerous protracted incubation periods and requires wash steps and generates vast quantities of radioactive waste. As a result, there is less usage as an HT method in drug screening programs eliminating shortlived background fluorescence.…”

“…22 However, in the presence of TNFα, the two labeled antibodies are moved into close proximity and this allows for a FRET transfer to occur between the donor and acceptor fluorophores. 14 This reaction produces a florescence signal at 665 nm. The increase of FRET levels is proportional to the concentration of TNFα.…”