Quantitation Of Human TNF-α With A QuantiGlo Assay From R&D Systems Using BioTek's Synergy 2

By Paul Held Ph. D., Senior Scientist, Applications Dept., BioTek Instruments, Inc.

The peptide TNF-α is a cytokine that plays a key role in inflammation and apoptosis. As such, quantitation of this cytokine from tissue culture lysates and from human serum and/or plasma samples under experimental and environmental conditions can provide important information regarding the regulation of these phenomenons. Here we describe the quantitation of TNF-α using a Synergy™ 2 Multi-Mode Microplate Reader form BioTek Instruments to measure the luminescence signal of a QuantiGlo kit from R&D Systems.

The cytokine TNF-a plays a key role in inflammation and apoptosis. TNF-α is synthesized as a 26 kD transmembrane protein, which is assembled as a noncovalently linked trimeric protein. A soluble 157 amino acid residue from the C-terminus of the protein is released by the activity of the metoloproteinase, TNF-α Converting Enzyme (TACE). A number of different cell types including B-cells, T-cells, macrophages, monocytes, mast cells, neutrophils and adipocytes are known to express TNF-α.

The human tumor necrosis factor alpha (TNF-α) assay is a sandwich ELISA. Microplates are coated using a monoclonal antibody (Mab) that recognizes an epitope on TNF-α. Standards and samples are pipetted into wells and any TNF- a present is captured and bound by the immobilized antibody. After washing away any unbound materials, an enzyme-linked polyclonal antibody conjugate specific for TNF-a is added to the wells. Following a second wash step to remove unbound antibody-enzyme conjugate, a luminol/peroxide substrate solution is added to the wells and light is produced in proportion to the amount of TNF-α present. A microplate reader capable of measuring luminescence is used to measure the intensity of the light produced.