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Characterization of a bioassay for detection of recombinant and native ovine interleukin‐5
Author(s) -
Bosward Katrina,
Emery David L,
McWaters Peter W,
Husband Alan J,
Bendixsen Tuan
Publication year - 1999
Publication title -
immunology and cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0818-9641
DOI - 10.1046/j.1440-1711.1999.00831.x
Subject(s) - recombinant dna , microbiology and biotechnology , biology , bioassay , sf9 , antiserum , interleukin 3 , immune system , monoclonal antibody , antibody , spodoptera , immunology , t cell , gene , biochemistry , interleukin 21 , genetics
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‐1β, IL‐2, IL‐3, IL‐6, IL‐8, stem cell factor (SCF) or IFN‐γ at doses from 0.01 to 1 μg/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 μL 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.