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.
Hope
J C
JC
Institute for Animal Health, Compton, Near Newbury, RG20 7NN, UK. Jayne.Hope@BBSRC.ac.uk
Kwong
L S
LS
Thom
M
M
Sopp
P
P
Mwangi
W
W
Brown
W C
WC
Palmer
G H
GH
Wattegedera
S
S
Entrican
G
G
Howard
C J
CJ
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