Chicken interleukin-26: characterization and antigen capture enzyme-linked immunosorbent assay

Interleukin-26, a cytokine belonging to the IL-10 family, is an inflammatory mediator in mammals and has been shown to modulate T cell proliferation and gene expression in avian species. Given the limited understanding of chIL-26's function at the protein level, our objective was to produce specific mAbs to chIL-26 and create an immunoassay to investigate its biological significance in chickens. We generated five anti-chIL-26 monoclonal antibody clones by using recombinant chIL-26 as an immunogen in mice. Western blot and indirect ELISA analyses confirmed that all five mAbs specifically recognize the recombinant chIL-26 protein. Using a pairing assay, we determined that the combination of capture antibody #10C7 and biotinylated detection antibody #8A5 was the most effective pair for a sandwich ELISA for chIL-26 detection. To further validate a newly developed antigen-capture ELISA chIL-26 detection, we first stimulated chicken macrophage cells with three agonists-Lipopolysaccharides, polyinosinic:polycytidylic acid, and Resquimod-848 at various concentrations. Second, qRT-PCR was used to confirm that LPS induced significant chIL-26 expression. The changes in chIL-26 production over time was monitored using a monoclonal antibody (mAb) combination (#10C7 and biotinylated #8A5). Antigen capture ELISA revealed a significant increase in IL-26 protein secretion, which peaked at 24 hours post-stimulation. This pattern mirrored the expression of chIL-26 mRNA, as detected by qRT-PCR. This assay was also successfully used to detect the changes in IL-26 levels in the serum of chickens infected with Eimeria maxima (E. maxima) and E. tenella. Compared to the uninfected chickens, IL-26 levels in E. maxima-infected chickens began to increase from 1-day post-infection (dpi), peaked at 3 dpi, and then rapidly decreased. In contrast, IL-26 levels in E. tenella-infected chickens peaked at 7 dpi. All newly produced mAbs specific for chIL-26 effectively neutralized the function of IL-26, as measured by cell proliferation assays and IL-1β expression using qRT-PCR analysis. These new anti-chIL-26 mAbs and the antigen-capture ELISA will be valuable tools for further dissecting the role of chIL-26 in poultry inflammatory responses and diseases.