Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were determined and a standard curve was established using recombinant chIFN-α. The sandwich ELISA effectively detected an increased IFN-α production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C), and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize its activity were identified. The newly developed antigen sandwich ELISA developed in this study will be a useful tool to monitor IFN-α production in chickens.

干扰素α（IFN-α）属于I型干扰素家族，可介导对病毒感染的早期先天免疫应答。在本研究中，我们开发了使用特异性小鼠单克隆抗体（mAbs）来测量鸡中IFN-α产生的夹心ELISA。酵母中表达的重组鸡IFN-α（chIFN-α）购自Kingfisher Biotech，并用于免疫小鼠。选择并鉴定了五个特异性识别鸡IFN-α抗原的mAb。对于夹心ELISA的开发，mAb用生物素标记，然后进行配对测试以鉴定最佳捕获和检测抗体。确定了两组小鼠抗chIFN-αmAb对，并使用重组chIFN-α建立了标准曲线。夹心酶联免疫吸附法有效地检测了多肌苷酸：聚胞苷酸（poly I：C）刺激的鸡巨噬细胞中IFN-α产生的增加，其最低可检测水平约为25 pg / mL。在禽胚胎成纤维细胞中鉴定了chIFN-α对水泡性口炎病毒的抗病毒活性，并鉴定了中和其活性的小鼠抗chIFN-αmAb。在这项研究中开发的新开发的抗原夹心ELISA将成为监测鸡中IFN-α产生的有用工具。