Development of Antigen-Capture Enzyme-linked Immunoassay for Chicken Interleukin-34.

Abstract

Interleukin-34 (IL-34), a recently identified cytokine, is known to share similar functions with macrophage colony-stimulating factor 1 (CSF-1). However, its function and active regions in avian species have not yet been fully understood. In this study, we report the functional characterization and immunomodulatory properties of chicken IL-34 (chIL-34) using a panel of newly developed anti-chIL-34 mouse monoclonal antibodies (mAbs). Five mAbs (3G11, 10A3, 11A7, 12A8, and 13C2) that specifically recognized recombinant chIL-34 protein were selected and characterized based on their binding activity toward recombinant chIL-34 protein via indirect ELISA and western blotting. A new chIL-34-specific antigen-capture sandwich ELISAs was developed using compatible mAb pairs (3G11 as the capture antibody and biotinylated-12A8 as the detection antibody) which have been identified through pairing assays. To validate the antigen-capture assay, we stimulated native chIL-34 production in the chicken HD11 macrophage cell line using three agonists, Lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (poly I:C), and Resquimod-848 (R-848) at varying concentrations (0.5, 1.0, and 2.0 μg/mL). The qRT-PCR confirmed that the significant expression of chIL-34 was induced by 2.0 μg/mL of LPS, which we selected as an agonist for further chIL-34 production in HD11 cell line. We further stimulated the HD11 cell line with 2.0 μg/mL LPS and monitored the resulting changes in chIL-34 production over time using a mAbs combination (12A8 and biotinylated-3G11). The IL-34 production peaked at 24 hours (h) post stimulation, followed by a decrease at 48 h. This pattern paralleled the expression of chIL-34 mRNA as detected by qRT-PCR. The changes in IL-34 levels in the serum of chickens infected with Eimeria maxima (E. maxima) and Eimeria tenella (E. tenella), measured using a combination of 12A8 and biotinylated-3G11, showed different patterns. Compared to uninfected chickens, IL-34 levels in E. maxima-infected chickens increased starting from 1-day post-infection (dpi), peaked between 3 and 5 dpi, and then rapidly decreased by 7 dpi, falling below the levels observed at 1 dpi. In contrast, IL-34 levels in E. tenella-infected chickens increased from 3 dpi and steadily rose at 5 and 7 dpi. All newly produced mAbs (3G11, 10A3, 11A7, 12A8, and 13C2) specific for chIL-34 effectively neutralized the function of IL-34, as measured by cell proliferation assays and IL-10 expression using qRT-PCR analysis. These new anti-chIL-34 mAbs and the antigen-capture ELISA will be valuable tools for both fundamental and applied research in avian immunology.