Development of detection methods for ruminant interleukin (IL)-4.
Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression 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, RG20 7NN, UK. Jayne.Hope@BBSRC.ac.uk
J C
JC
Hope
L S
LS
Kwong
M
M
Thom
P
P
Sopp
W
W
Mwangi
W C
WC
Brown
G H
GH
Palmer
S
S
Wattegedera
G
G
Entrican
C J
CJ
Howard
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000276:immunology; D019556:COS Cells; D002417:Cattle / Q000276:immunology*; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D015847:Interleukin-4 / Q000032:analysis* / Q000276:immunology*; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D015847:Interleukin-4