Development of detection methods for ruminant interleukin (IL)-12.
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.
Institute for Animal Health, Compton, Near Newbury, UK. Jayne.Hope@BBSRC.ac.uk
J C
JC
Hope
L S
LS
Kwong
G
G
Entrican
Sean
S
Wattegedera
H M
HM
Vordermeier
P
P
Sopp
C J
CJ
Howard
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019556:COS Cells; D002417:Cattle / Q000276:immunology*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D003713:Dendritic Cells / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D018664:Interleukin-12 / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D012756:Sheep
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D018664:Interleukin-12