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

Authors: LEE, YOUNGSUB - US Department Of Agriculture (USDA); KIM, WOO HYUN - Gyeongsang National University; NAM, HYOYOUN - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2024
Publication Date: 8/1/2024
Citation: Lee, Y., Kim, W., Nam, H., Lillehoj, H.S. 2024. Differential detection of chicken heterodimeric cytokines, interleukin 12 and 23 using their subunit-specific mouse monoclonal antibodies. Poultry Science. VOL.103/ 103872. https://doi.org/10.1016/j.psj.2024.103872.
DOI: https://doi.org/10.1016/j.psj.2024.103872

Interpretive Summary: Cytokines are soluble mediators of immune response and ability to measure their production is critical for the assessment of host disease response. However, there is limited information on cytokines in chickens and no immunoassays for measuring cytokines, especially for IL-12 and IL-23. In this paper, ARS scientists developed novel quantitative immunoassays to measure chicken IL-12 and IL-23 which play crucial roles in both protective immunity against intracellular pathogens. In this paper, we developed novel immunoassays using pairs of mouse monoclonal antibodies which are specific against chicken IL-12 and IL-23 subunits. These immunoassays were validated for their specificities against chicken IL-12 and IL-23 using immune serum from Eimeria-infected chickens. Availability of antigen-specific immunoassays specifically designed for poultry IL-12 and IL-23 will not only provide the advantage of quantifying these two cytokines at the protein level in chickens but will also facilitate investigations into avian IL-12 and IL-23 immunobiology.

Technical Abstract: Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function. IL-12 and IL-23 share a common p40 subunit, but differ in their p35 and p19 subunits, respectively. This difference in subunit composition results in distinct signaling pathways and biological functions for IL-12 and IL-23. Here, we report the functional characterization and immunomodulatory properties of chicken IL-12 and IL-23 using the panels of newly developed mouse anti-IL-12p40, IL-12p35-a and IL-23p19 monoclonal antibodies (mAbs). Western blot and indirect ELISA analysis demonstrated that the anti-chicken IL-12p40 mAbs (chIL-12p40; #10G10F4 and #10D8G2) bound to both recombinant proteins (IL-12 and IL-23), the anti-chicken IL-12p35 mAb (chIL-12p35; #2F1) specifically recognized recombinant IL-12, and the anti-chicken IL-23p19 mAb (chIL-23p19; #15A3) exhibited specificity for recombinant IL-23, without any cross-reactivity. Two ELISAs detecting specific chicken IL-12 (#10G10F4 and #2F1) or IL-23 (#10D8G2 and #15A3) were developed using newly developed mAb combinations, #10G10F4/ #2F1 and #10D8G2/#15A3 for Il-12 and IL-23, respectively, identified through a pairing assay. The levels of IL-12 and IL-23 in Resiquimod-848 stimulated-HD11 chicken macrophage cells were monitored over time using antigen-capture sandwich ELISA developed in this study. Furthermore, the levels of chicken IL-12 and IL-23 in the circulation of Eimeria species-infected chickens were determined. Notably, the anti-chIL-12p40 mAbs (#10G10F4 and #10D8G2) neutralized the function of both chIL-12 and chIL-23 proteins, which share the p40 subunit, while the anti-chIL-23p19 mAb (#15A3) specifically neutralized chIL-23 protein in HD11 cells in vitro. The anti-chIL-12p35 mAb (#2F1), which is specific to the p35 subunit of IL-12, showed a slight neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of two newly developed antigen-capture immunoassays for chIL-12 and chIL-23 which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These mAbs for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.