A sensitive new bioassay for tumor necrosis factor

Details

• Personal Author:
  Kuschner WG ; Lewis, Daniel M. ; Shahan TA ; Siegel, Paul D. ; Sorenson WG
• Description:
  An improved tumor necrosis factor (TNF) bioassay using an isolated subclone from the murine fibroblastoid NCTC-clone-929 cell line and a new fluorescence indicator system for detecting target cell viability was described. The NCTC-clone-929 cell line was cultured and cloned. Target cells were released from attachment, centrifuged, counted, and diluted to a density of 2x10(4) cells per well before a 24 hour incubation with TNF-alpha standards or unknowns. After removal of medium and sample, the cells were incubated with 10% Alamar-blue for 12 hours. Alamar-blue fluorescence was read on a fluorescence 96 well plate counter at 560 to 590 nanometers. Neutral-red and crystal-violet assays were performed for comparison with the Alamar-blue bioassay. Cells harvested from CD-rats by bronchoalveolar lavage were isolated and then incubated with varying concentrations of lipopolysaccharide (LPS) for the detection of TNF activity in LPS macrophage conditioned medium. A TNF hypersensitive subclone A12 from the NCTC- clone-929 was isolated and found to be sensitive to as little as 68+/-2.5 femtograms/milliliter of TNF-alpha. Incubation of the cells in different sera and varying serum concentrations did not indicate any significant differences between sera, but revealed a significant reduction in sensitivity if less than 15% serum was used. A comparison of crystal-violet and neutral-red as indicators of viability showed that the Alamar-blue bioassay was three and four times more sensitive, respectively. TNF activity was detected with 3.0 nanograms/milliliter LPS. The authors conclude that the Alamar- blue assay employing the NCTC-clone-929 subclone A12 is effective in the quantitation of low levels of TNF, particularly when a suitable immunoassay is unavailable. [Description provided by NIOSH]
• Subjects:
  Biochemistry Biological Assay Cells, Cultured Histocytochemistry Immune System Occupational Health Safety
• Keywords:
  Bioassays Biochemical-indicators Cell-culture-techniques Cell-cultures Cytochemistry Immunochemistry Mammalian-cells NIOSH-Author
• ISSN:
  0022-1759
• Document Type:
  Text
• Genre:
  Journal Article
• Place as Subject:
  OSHA Region 3 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  DRDS - Division of Respiratory Disease Studies
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  181-187
• Volume:
  175
• Issue:
  2
• NIOSHTIC Number:
  nn:00225362
• Citation:
  J Immunol Meth 1994 Oct; 175(2):181-187
• Federal Fiscal Year:
  1995
• Peer Reviewed:
  True
• Source Full Name:
  Journal of Immunological Methods
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:0afbcba031e6f711f7d21195f16cfa0e4639d39b0d38250538fd5818676876a047bca7276e5a2ccd79623da49ef70147ce80ebf4d5f37c571456ac17bd9e3a45
• Download URL:
  https://stacks.cdc.gov/view/cdc/204056/cdc_204056_DS1.pdf
• File Type:
  [PDF - 500.00 KB ]