TNF assay system

The bead-based technology works not only for inflammatory mediators that have diagnostic/prognostic valne, bnt also for others (e.g., C-reactive protein, IL-6). This can make bead-based systems even more powerful. Kofoed et al. combined in-house and commercially available kits and used bead-based Luminex systems to assay biomarkers of potential interest in EDTA-plasma samples (70). A 3-plex assay for suPAR, sTREM-1, MIF was added to a commercially available human cytokine panel, IL-1P, IL-6, IL-8, GM-CSF, and TNF-a. Compared to healthy controls, all eight analytes were significantly higher in plasma from bacterial sepsis patients. 

Endotoxicity results from the interaction of a bacterial cell envelope component (e.g., LPS or PG with a cell surface receptor constituting part of the nonspecific immune system, (i.e., a toll-like receptor on white blood cells). This results in the production of cytokines [e.g., interleukin 1 (IL-1) or tumor necrosis factor (TNF)] as part of an intracellular enzyme cascade which can cause severe tissue injury. Bioassays or immunoassays can be used to detect such reactions respectively. As noted above the most widely used bioassay is the LAL assay. A lysate of amoebo-cytes of the horseshoe crab (Limulus) contains an enzymatic clotting cascade which is activated by extremely low levels of LPS (nanogram levels or lower). There are variants of this assay that can detect PG, but they are not as widely used. As noted above, other bioassays employ cultured cell lines that respond to LPS or PG, respectively. Unfortunately bioassays are highly amenable to false positives (from the presence of cross-reactive substances) or false negatives from inhibition (by contaminants present in the sample) [10]. A detailed discussion of these assays is beyond the scope of this chapter and has been reviewed elsewhere [1]. 

AlphaScreen applications include assays for enzymes such as protein kinases and proteases, immunoassays such as cAMP detection, and protein-protein and protein-DNA interactions. A recent literature example is a comparison of AlphaS-creen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. In this comparison, the AlphaScreen system showed the highest sensitivity and the broadest dynamic range [149]. Another recent publication concerns a high-throughput binding assay for a TNF receptor [175]. 

Approximately 2.5-5 X 10 cells were seeded into 24-well plates and allowed to adhere overnight. Dulbecco s modified Eagle s media (phenol red free) with supplements were added to the cells (Mitrovic et ah, 1994). The cells received no treatment, received interferon-y (IFN-y) (500 U/ml) and interleukin-1/8 (IL-1/3) (300 U/ml), or were pretreated with pentoxifylline (PTX) (1 mg/ml for 24 hr) before stimulation with IFN-y and IL-lj3. After 4-7 days of stimulation, cell supernatants were removed and stored at —40°C. Samples were thawed, and soluble TNF-Rl and TNF-R2 levels were assayed using enzyme-linked immunosorbent assay kits [human soluble tumor necrosis factor receptor type 1 (sTNF-Rl) and sTNF-R2 Quantikine kits, R D Systems, Minneapolis, MN]. For the sTNF-Rl kit the minimum detectable dose is 1 pg/ral. For the TNF-R2 kit the minimum detectable dose is 0.5 pg/ml. PTX was provided courtesy of Hoechst-Roussel Pharmaceuticals (Somerville, NJ). Values are expressed as means SD. 

In total, we have 24 different potential targets for the 9 GRAS molecules. For the sake of cost effectiveness and efficiency, we chose to employ a RAW 264.7 cell model—as described in the Materials and Methods section— to validate experimentally the putative anti-inflatmnatory effects of our compounds. This high-content assay allows one to measure several important inflammation-related parameters, such as NO, TNF-o, IL-1, IL-6, and PGE-2 activities. In our test assays, we also included compounds such as resveratrol as references, since it is known to have anti-inflammatoiy effects in this cell-based screening system as well as in other in vitro and in vivo models. Compounds were evaluated according to their maximum nontoxic concentration according to MTS assay (Table 4.4). 

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