Differential detection of chicken heterodimeric cytokines, interleukin-12 and -23 using specific monoclonal antibodies to their subunits

Authors: KIM, WOOHYUN - US Department Of Agriculture (USDA); LIM, YEASEUL - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/1/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Objective: No immune tools to detect at protein level chicken interleukin (IL)-12 or IL-23 which are heterodimeric cytokines sharing p40 subunit are available at present. In this study, we describe several mouse monoclonal antibodies which are specific to their subunits and sandwich ELISAs to analyze chicken IL-12 or IL-23. Methods: Three recombinant subunits, p19, p35, and p40 were expressed from yeast and E. coli and used as immunogens to develop mouse monoclonal antibodies (mAbs) for each subunit. Mice were immunized with each recombinant protein and hybridoma were generated. After screening of hybridoma by ELISA, mAbs were purified from hybridoma supernatant and their specificities to each subunit were verified using various assays including Western blot and immunocytochemistry. Sandwich ELISAs were established for IL-12 and IL-23 using all possible paring of p35 and p40 mAbs, or p19 and p40 mAbs, respectively. To determine if these mAbs could bind to native proteins, primary chicken lymphocytes were activated, and supernatant were subjected to further assays. The neutralization effect of these mAbs to inhibit the activity of mammalian expressed IL-12 or IL-23 was also investigated. Results: At least 10 ELISA-positive and single-cloned hybridomas for each subunit were produced. Several mAbs showed reactivity in Western blot and/or immunocytochemistry. Furthermore, mAbs that optimally detect and capture IL-12 (p40/p35 as capture/detection antibody) and IL-23 (p40/p19) were selected for sandwich ELISA. Using these systems, we found the expression of IL-12 and IL-23 protein in activated lymphocytes. We also found neutralizing mAbs to exhibit anti-IL-12 or -IL-23 activity by inhibiting the induction of IFN-' and IL-17A in chicken T cells. Conclusions: In this study, we have generated novel sensitive ELISA system to analyze specific chicken IL-12 and IL-23 at protein levels, and identified several mAbs that have neutralization activity against chicken IL-12 and IL-23. We believe these mAbs and ELISAs are valuable immune tools for poultry research to obtain a new insight to on chicken chicken T cell immunity. Keywords: chicken, IL-12, IL-23, monoclonal antibody