Animal Bioscience and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA.
College of Veterinary Medicine & Institute of Animal Medicine, Gyeongsang National University, Jinju 52828, South Korea.
Poult Sci. 2024 Aug;103(8):103872. doi: 10.1016/j.psj.2024.103872. Epub 2024 May 16.
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-α 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 maxima (E. maxima) and Eimeria tenella (E. tenella)-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 partial neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of 2 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.
白细胞介素-23（IL-23）是最近发现的白细胞介素-12 家族异二聚体细胞因子成员，在调节辅助性 T 细胞功能方面发挥着关键作用。IL-12 和 IL-23 具有共同的 p40 亚基，但在其 p35 和 p19 亚基方面存在差异。亚基组成的这种差异导致了 IL-12 和 IL-23 具有不同的信号通路和生物学功能。在这里，我们使用新开发的抗鸡白细胞介素-12p40、IL-12p35-α和 IL-23p19 单克隆抗体（mAb）的面板，报告了鸡白细胞介素-12 和 IL-23 的功能特征和免疫调节特性。Western blot 和间接 ELISA 分析表明，抗鸡白细胞介素-12p40 mAb（chIL-12p40；#10G10F4 和 #10D8G2）与重组蛋白（IL-12 和 IL-23）结合，抗鸡白细胞介素-12p35 mAb（chIL-12p35；#2F1）特异性识别重组 IL-12，抗鸡白细胞介素-23p19 mAb（chIL-23p19；#15A3）对重组 IL-23 表现出特异性，没有任何交叉反应。使用新开发的 mAb 组合#10G10F4/ #2F1 和 #10D8G2/ #15A3 开发了两种特异性检测鸡白细胞介素-12（#10G10F4 和 #2F1）或白细胞介素-23（#10D8G2 和 #15A3）的 ELISA，这些 mAb 通过配对试验确定，分别为 IL-12 和 IL-23。使用本研究中开发的抗原捕获夹心 ELISA 监测 Resiquimod-848 刺激的 HD11 鸡巨噬细胞中白细胞介素-12 和白细胞介素-23 的水平随时间的变化。此外，还测定了鸡白细胞介素-12 和白细胞介素-23 在感染最大艾美耳球虫（E. maxima）和柔嫩艾美耳球虫（E. tenella）鸡中的循环水平。值得注意的是，抗鸡白细胞介素-12p40 mAb（#10G10F4 和 #10D8G2）中和了共享 p40 亚基的鸡白细胞介素-12 和鸡白细胞介素-23 蛋白的功能，而抗鸡白细胞介素-23p19 mAb（#15A3）在体外特异性中和了 HD11 细胞中的鸡白细胞介素-23 蛋白。特异性针对白细胞介素-12 p35 亚基的抗鸡白细胞介素-12p35 mAb（#2F1）对鸡白细胞介素-12 蛋白表现出部分中和作用。总的来说，我们的研究验证了两种新开发的鸡白细胞介素-12 和白细胞介素-23 抗原捕获免疫测定的特异性和意义，这将扩展我们对白细胞介素-12 和白细胞介素-23 的功能特征及其在正常和患病鸡中的关联的理解。这些针对每个亚基的 mAb，抗鸡白细胞介素-12p35、抗鸡白细胞介素-12p40 和抗鸡白细胞介素-23p19，将作为有价值的免疫试剂，阐明宿主对疾病发病机制的免疫反应，无论是在禽类的基础研究还是应用研究中。
