Development of detection methods for ruminant interleukin (IL)-12

Jayne Hope, L.S. Kwong, Gary Entrican, Sean Wattegedera, H.M. Vordermeier, Paul Sopp, Chris J Howard

Research output: Contribution to journalArticlepeer-review

Abstract

Recombinant bovine IL-12 (rbo IL-12) was transiently expressed in COS-7 cells and shown to upregulate the synthesis of IFNgamma by bovine cells stimulated with a suboptimal concentration of mitogen in vitro. Mice were immunised with a plasmid encoding rbo IL-12 and boosted with rbo IL-12 and a number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-12 in an ELISA. Some of these mAb neutralised the ability of rbo IL-12 to induce IFNgamma synthesis by bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-12 by ELISA and a luminometric detection method was applied to the ELISA making it more sensitive. Using this method native bovine IL-12 was detected in supernatants of dendritic cells (DC) cultured in vitro with a synthetic lipopeptide known to stimulate secretion of IL-12 by human DC. The ELISA was also able to detect recombinant ovine IL-12 and, less effectively, recombinant human IL-12. In contrast, bovine IL-12 was not detected by a commercial human IL-12 ELISA kit. Intracytoplasmic IL-12 was detected in bovine DC using the antibodies described herein. The ability to detect ruminant IL-12 by three methods: ELISA, bioassay with neutralising mAb and cytoplasmic staining, will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.
Original languageEnglish
Pages (from-to)117-26
Number of pages10
JournalJournal of Immunological Methods
Volume266
Issue number1-2
Publication statusPublished - 1 Aug 2002

Keywords

  • Animals
  • Antibodies, Monoclonal/immunology
  • COS Cells
  • Cattle/immunology
  • Cells, Cultured
  • Cytoplasm/chemistry
  • Dendritic Cells/immunology
  • Enzyme-Linked Immunosorbent Assay/methods
  • Humans
  • Interleukin-12/analysis
  • Interleukin-12/genetics
  • Interleukin-12/immunology
  • Interleukin-12/pharmacology
  • Mice
  • Mice, Inbred BALB C
  • Neutralization Tests
  • Recombinant Proteins/analysis
  • Recombinant Proteins/pharmacology
  • Sheep

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