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.
Hope
J C
JC
Institute for Animal Health, Compton, Near Newbury, UK. Jayne.Hope@BBSRC.ac.uk
Kwong
L S
LS
Entrican
G
G
Wattegedera
Sean
S
Vordermeier
H M
HM
Sopp
P
P
Howard
C J
CJ
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