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.

干扰素 (IFN)-κ 是一种 I 型干扰素，在抗病毒防御和宿主免疫反应中起核心作用。与哺乳动物对应物相比，I 型干扰素在鸡中的功能尚未明确定义。在本研究中，我们使用新开发的小鼠单克隆抗体 (mAb) 开发了一种抗原捕获 ELISA，该抗体对鸡 IFN-κ (chIFN-κ) 具有特异性，并表明该 ELISA 可以测量巨噬细胞活化过程中天然 chIFN-κ 的产生通过聚肌苷：聚胞苷酸（聚 I：C）。在大肠杆菌中表达的重组鸡干扰素-κ用于免疫小鼠。通过间接 ELISA 和蛋白质印迹，根据它们对 chIFN-κ 的特异性和结合活性，选择并表征了五种特异性识别 chIFN-κ 的 mAb。为了开发鸡 IFN-κ 的捕获 ELISA，通过配对分析确定了两组最佳捕获和检测 mAb 组合。为了验证抗原捕获测定，使用 poly I:C 在鸡 HD11 巨噬细胞中诱导天然 IFN-κ 的产生。此外，qRT-PCR 用于确认 24 和 48 小时 HD11 细胞中 IFN-κ 的转录水平表达。抗 chIFN-κ mAb 的中和作用是基于其阻断重组 chIFN-κ 蛋白刺激的 DF-1 成纤维细胞中 IFN 刺激基因 (ISG) 诱导的能力来评估的。所有五种 mAb 均以剂量依赖性方式阻断 ISG 的 mRNA 表达。我们的结果验证了这些新开发的 mAb 用于检测天然鸡 IFN-κ 的特异性和实用性。这种新颖的抗原捕获 ELISA 将成为涉及正常和疾病状态下 IFN-κ 的基础和应用研究的宝贵工具。