|LEE, YS - Chung-Ang University
|KIM, WH - Chung-Ang University
|LEE, SJ - Kangwon National University
Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2020
Publication Date: 11/1/2020
Citation: Lee, Y., Kim, W., Lee, S., Lillehoj, H.S. 2020. Development of antigen capture ELISA to detect interferon-alpha (IFN-a) using monoclonal antibodies in chicken. Poultry Science. https://doi.org/10.1016/j.vetimm.2020.110124.
Interpretive Summary: Understanding how the Innate immune system works in defense against infections is critical first step to develop a logical preventive strategy against various diseases. Innate immunity is carried out by different types of cells and various soluble factors called cytokines and chemokines which are secreted by innate immune cells. Immunoregulatory cytokines such as interleukin (IL)-5, -10, and -13 mediate host defense by activating immune cells including natural killer (NK) cells and macrophages. In viral infection, interferon alpha and beta (IFN-a/ß) are critical in defense against them and can be induced from the early stages of virus infection. There is a limited information on poultry innate immune system and immune mediators. Although the mechanism of type ' INFs in mammalian is well established, their role in immune response against viral infection in poultry is lacking because of our inability to detect them. Therefore, ARS scientists developed new immune reagents that can detect chicken cytokines in collaboration with scientists at a South Korean university to better understand their function in disease process. This new enzyme-linked immunoassay (ELISA) specifically detects chicken IFN-a using mouse monoclonal antibodies (mAbs) and this will be useful for field veterinarians and scientists conducting basic and applied research in poultry.
Technical Abstract: Interferon alpha (IFN-a) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed capture ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-a production in chicken. Recombinant chicken IFN-a was cloned, expressed in yeast, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-a antigen were selected and characterized. For capture ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify capture and detection antibodies. Two sets of mAb pairs were determined and a standard curve was established using recombinant chicken IFN-a (ChIFN-a). The capture ELISA effectively detected an increased IFN-a production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C). The anti-viral activity of chIFN-a against vesicular stomatitis virus was identified in avian embryonic fibroblast and IFN-a mAbs could neutralize its activity. The newly developed antigen capture ELISA developed in this study could be a useful tool to monitor native INF-a production in chicken.