Production of antibodies to canine IL-1β and canine TNF to assess the role of proinflammatory cytokines

IL-1 and TNF are important proinflammatory cytokines implicated in both antimicrobial host defense and pathogenesis of diseases with an immune-mediated and/or inflammatory component. Respective studies in the dog have been hampered by the unavailability of reagents allowing the specific measurement of canine cytokine proteins and the effect of canine cytokine neutralization by Ab. Starting with recombinant canine (rcan) IL-1β and rcanTNF, four polyclonal antisera and 22 mAb specific for rcanIL-1β and rcanTNF were generated. Their usefulness in neutralization assays was determined. Using cytokine-containing supernatants of canine cells in bioassays, polyclonal antisera neutralized either canine IL-1β or TNF. TNF was also neutralized by three antibodies developed in this study and one commercial mAb. The usefulness of monoclonal and polyclonal Ab in canine cytokine-specific Ab capture ELISAʹs was assessed. This resulted in the identification of a commercial mAb combination and one pair developed in this study allowing low levels of TNF to be detected by antibody capture ELISA. The detection limit was 141 pg/ml rcanTNF for both combinations. Using rcanIL-1β as an antigen allowed the detection of lower concentrations of rcanIL-1β (20 pg/ml, on the average) by a pair of polyclonal antisera than when monoclonals were used. By using such IL-1β-specific and TNF-specific ELISAʹs, the respective cytokines were detected in supernatants of canine PBMC stimulated with LPS or heat-killed Listeria monocytogenes and interferon-γ combined. Thus, monoclonal and polyclonal reagents were identified allowing the quantitation of canine IL-1β and TNF production in vitro, and the neutralization of these cytokines.