Fig. (1) Principle of HTRF-based TNF-α assay. The assay was designed using time resolved fluorescence resonance energy transfer (TRFRET) technology. In this assay, two anti-TNF-α antibodies are labeled with europium Cryptate and XL665, respectively. In the absence of TNF-α, there is no FRET between these two labeled antibodies, which produces fluorescent signal at 615 nm (Fig. 1A). When TNF-α is present, these two labeled antibodies are brought into close proximity of each other, which allows FRET to occur between the two fluorophores, europium Cryptate and XL665 (Fig. 1B). This reaction produces a fluorescent signal at 665 nm. The increase of FRET levels is proportional to the concentrations of TNF-α.