Characterization of a bioassay for detection of recombinant and native ovine interleukin-5.
In order to analyse Th2-type immune responses in sheep by the assay of interleukin (IL)-5 in biological fluids, the ovine IL-5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC-OV-IL-5 from the supernatant fluid. The ovine IL-5 was biologically active in a bioassay using IL-5-dependent Baf cells, which have been used previously to specifically detect human IL-5. The specificity of Baf cells for ovine IL-5 was examined by two methods. First, Baf cells only proliferated in response to BAC-OV-IL-5 and did not respond to addition of recombinant ovine cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1beta, IL-2, IL-3, IL-6, IL-8, stem cell factor (SCF) or IFN-gamma at doses from 0.01 to 1 microg/well. Second, the rat monoclonal antibody to murine IL-5, TRFK-5, neutralized murine, but not ovine, IL-5. However, rabbit antisera to BAC-OV-IL-5 neutralized murine and ovine recombinant IL-5 and abolished responses of Baf cells to IL-5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 microL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL-5 also extends to ovine IL-5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.
Bosward
K
K
Department of Veterinary Anatomy and Pathology, The University of Sydney, New South Wales, Australia.
Emery
D L
DL
McWaters
P W
PW
Husband
A J
AJ
Bendixsen
T
T
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D001681:Biological Assay / Q000379:methods* / Q000706:statistics & numerical data; D002460:Cell Line; D003001:Cloning, Molecular; D016207:Cytokines / Q000302:isolation & purification; D006801:Humans; D015848:Interleukin-5 / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D008213:Lymphocyte Activation; D051379:Mice; D009500:Neutralization Tests; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012756:Sheep; D018411:Spodoptera
D016207:Cytokines; D015848:Interleukin-5; D011994:Recombinant Proteins