A sensitive new bioassay for tumor necrosis factor.
Tumor necrosis factor is an important cytokine involved in inflammation and assay of this cytokine in biological fluids may be important in the understanding of several disease processes. This report describes an improved TNF bioassay employing a newly isolated subclone of the cell line NCTC-clone 929 as well as a novel fluorescence indicator system for detecting viability of the target cells. The limit of detection for the TNF hypersensitive cell line with this fluorescence viability assay was 68 +/- 2.5 fg/ml, which is approximately 3 x more sensitive than the parental clone and approximately 10 x more sensitive than that reported by Branch et al. (1991) using the neutral red indicator system. The hypersensitivity of the clone gradually declined over a 45-day period and at regular intervals new cells were cultivated from frozen stocks. Two different serum sources, bovine fetal serum and horse serum, and four different serum concentrations (5, 10, 15, 20%) were evaluated to optimize sensitivity. No difference was found between serum sources but sensitivity was significantly reduced if < 15% serum was used.
Shahan
T A
TA
Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Morgantown, WV 26505.
Siegel
P D
PD
Sorenson
W G
WG
Kuschner
W G
WG
Lewis
D M
DM
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002460:Cell Line; D002999:Clone Cells; D004396:Coloring Agents; D003601:Cytotoxicity Tests, Immunologic; D005840:Gentian Violet; D008070:Lipopolysaccharides; D016676:Macrophages, Alveolar / Q000276:immunology; D051379:Mice; D009499:Neutral Red; D010078:Oxazines; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*; D014966:Xanthenes
D004396:Coloring Agents; D008070:Lipopolysaccharides; D010078:Oxazines; D014409:Tumor Necrosis Factor-alpha; D014966:Xanthenes; C005843:resazurin; D009499:Neutral Red; D005840:Gentian Violet