A specific and reliable bioassay for the detection of femtomolar levels of human and murine tumor necrosis factors

A reliable, highly sensitive, cytolytic bioassay for the quantitation of both human and murine tumor necrosis factor (TNF) is described. The assay is 2–180-fold more sensitive than other currently described bio- or immunoassays (limits of detection: 500 fg/ml (29 fmol/1) human TNF-α, 200 fg/ml (12 fmol/1) murine TNF-α and 130 fg/ml (7 fmol/1) human TNF-β). The assay, which uses L929-8, a newly isolated subclone of the murine fibroblastoid cell line L929, detects human TNF-α approximately 180-fold more sensitively than previously described L929 subclone assays. Maximum sensitivity is obtained by preincubating L929-8 cells at 37°C with 2 μg/ml actinomycin D (1–2 h), then culturing with TNF at 40°C for 20 h in medium containing high serum (15% FBS). Relative viable cell content in 96-well microtiter plates is determined colorimetrically by uptake of the non-carcinogenic dye neutral red. Other cytokines have no effect, either alone or in combination with TNF. Cytokines tested were IL-1 through IL-6, GM-CSF, G-CSF, CSF-1, LIF, TGB-β, NGF, Epo or IFN-γ. LPS, PGE 2, dexamethasone and cyclosporin A, also have no effect, either alone or in combination with TNF. L929-8 cells maintain the above sensitivity to TNF for at least 4 months in continuous culture. Thus, the assay allows rapid, inexpensive, reliable and specific quantitation of rodent and human TNFs. Its very high sensitivity should allow accurate detection of biologically active TNF in biological fluids such as human serum.