Detection of tumor necrosis factor α from porcine alveolar macrophages using an L929 fibroblast bioassay

Mary Jo Baarsch, Michael J. Wannemuehler, Thomas W Molitor, Michael P Murtaugh

Research output: Contribution to journalArticle

64 Scopus citations

Abstract

Tumor necrosis factor plays a central role in the mediation of the pathophysiological sequelae of infection and inflammation in animals and humans. The elucidation of its role in respiratory disease of swine has not been investigated, due in part to the lack of a sensitive and specific quantitative assay for its presence in tissue and fluid samples. Here we describe the detection of porcine tumor necrosis factor utilizing L929 murine fibroblast cells and characterize various parameters affecting assay sensitivity. Plating cell density and length of exposure time to test supernatants were the most critical factors. Using standard assay conditions as described here, porcine tumor necrosis factor was detected in alveolar macrophage conditioned media diluted more than 400-fold. Specificity of the assay for porcine tumor necrosis factor was shown by inhibition of cytotoxicity with neutralizing polyclonal antibodies for human recombinant tumor necrosis factor. Furthermore, comparisons of bioactivity with tumor necrosis factor mRNA levels from lipopolysaccharide-stimulated porcine alveolar macrophages indicated that the L929 bioassay was specific for porcine tumor necrosis factor.

Original languageEnglish (US)
Pages (from-to)15-22
Number of pages8
JournalJournal of Immunological Methods
Volume140
Issue number1
DOIs
StatePublished - Jun 24 1991

Keywords

  • 929 bioassay
  • Alveolar macrophage, porcine
  • Respiratory disease, swine
  • Tumor necrosis factor

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