Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Abstract

Interferon alpha (IFN-alpha) 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-alpha production in chickens. Recombinant chicken IFN-alpha (chIFN-alpha) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-alpha 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-alpha mAb pairs were determined and a standard curve was established using recombinant chIFN-alpha. The sandwich ELISA effectively detected an increased IFN-alpha 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-alpha against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-alpha 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-alpha production in chickens.