Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA

A purified recombinant ovine (rOv) interleukin-6 (LL-6) was used to generat e specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit anti sera to this cytokine. From the 31 initial hybridoma cell Lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as jud ged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blott ing. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytok ines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and We stern blotting. In contrast, the polyclonal antibodies only cross-reacted w ith bovine IL-6 and not with either of the human or monkey homologues. By c ombining a mAb with the polyclonal antisera a sensitive, IL-6-specific, cap ture ELISA was developed that had a sensitivity of 150 pg/ml. This detectio n system was unequivocally validated by demonstrating that native OvIL-6 co uld be detected in efferent lymph draining from a stimulated popliteal lymp h node. In addition, one of the mAbs was shown to allow the detection of OV IL-6 by intracellular cytokine staining and flow cytometry. (C) 2000 Elsevi er Science B.V. All rights reserved.