Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA.
A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.
McWaters
P
P
CRC for Vaccine Technology Unit, CSIRO Animal Health, Parkville, Vic., Australia.
Hurst
L
L
Chaplin
P J
PJ
Collins
R A
RA
Wood
P R
PR
Scheerlinck
J P
JP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D015153:Blotting, Western / Q000662:veterinary; D019556:COS Cells; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D006825:Hybridomas / Q000737:chemistry / Q000276:immunology; D007106:Immune Sera / Q000096:biosynthesis; D015850:Interleukin-6 / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity; D012756:Sheep / Q000276:immunology*; D014162:Transfection / Q000276:immunology
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D015850:Interleukin-6; D011994:Recombinant Proteins