Interferon (IFN)-κ is a type I IFN that plays a central role in anti-viral defense and host immune response. The functions of type I IFNs have not been clearly defined in chickens compared to those of their mammalian counterparts. In this study, we developed an antigen-capture ELISA using newly developed mouse monoclonal antibodies (mAbs) which are specific for chicken IFN-κ (chIFN-κ) and showed that this ELISA can measure native chIFN-κ production during the activation of macrophages by polyinosinic:polycytidylic acid (poly I:C). The recombinant chicken IFN-κ expressed in Escherichia coli was used to immunize mice. Five mAbs that specifically recognized chIFN-κ were selected and characterized based on their specificity and binding activity toward chIFN-κ via indirect ELISA and western blotting. To develop a capture ELISA for chicken IFN-κ, two sets of the best capture and detection mAbs combinations were identified via pairing assays. To validate the antigen-capture assay, the production of native IFN-κ was induced in chicken HD11 macrophages using poly I:C. Furthermore, qRT-PCR was used to confirm the transcript-level expression of IFN-κ in HD11 cells at 24 and 48 h. The neutralizing effects of anti-chIFN-κ mAbs were evaluated based on their ability to block the induction of IFN-stimulated genes (ISGs) in DF-1 fibroblast cells stimulated with recombinant chIFN-κ proteins. All five mAbs blocked the mRNA expression of ISGs in a dose-dependent manner. Our results validate the specificity and utility of these newly developed mAbs for the detection of native chicken IFN-κ. This novel antigen-capture ELISA will be a valuable tool for fundamental and applied research involving IFN-κ in the normal and diseased states.
Keywords: Capture ELISA; IFN-κ; Monoclonal antibody; Type Ι IFNs; mRNA.
Published by Elsevier Ltd.