Development of antigen-capture ELISA to analyze the immunological functions of chicken interferon-kappa using specific monoclonal antibodies

Authors: LEE, YOUNGSUB - US Department Of Agriculture (USDA); LU, MINGMIN - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/8/2021
Publication Date: 7/13/2021
Citation: Lee, Y., Lu, M., Lillehoj, H.S. 2021. Development of antigen-capture ELISA to analyze the immunological functions of chicken interferon-kappa using specific monoclonal antibodies. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2021.104204.
DOI: https://doi.org/10.1016/j.dci.2021.104204

Interpretive Summary: Interferons (IFNs) are the most important chemokine involved in innate and adaptive host defense against viral diseases since the main function of IFNs is to inhibit viral replication. One type of IFNs, IFN-k, has recently been identified in chickens and it markedly inhibited the replication of RNA viruses, such as the avian influenza virus and Newcastle disease virus. In this report, ARS scientists developed a new antigen-capture ELISA which selectively identify chicken IFN-k in biological samples. The recombinant chicken IFN-k protein was used to immunize mice and 5 mouse monoclonal antibodies were selected. To develop a capture ELISA for chicken IFN-k, two sets of the best capture and detection mAb combinations were identified via pairing assays. This antigen-capture assay was used to measure IFN-k production following the induction of IFN-stimulated genes (ISGs) in DF-1 cells. The results validated the specificity of these new monoclonal antibodies for the detection of native chicken IFN-k. This novel antigen-capture ELISA will be used to detect native chicken IFN-k in biological samples to study the mechanism of innate protection against infection with chicken IFN-k.

Technical Abstract: Interferon IFN-k 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 chicken IFN-k-specific mouse monoclonal antibodies (mAbs), and investigated the role of chicken IFN-k in disease progression. The recombinant chicken IFN-k expressed in Escherichia coli was used to immunize mice. Five mAbs that specifically recognized chIFN-k were selected and characterized, based on their specificity and binding activity toward chIFN-k via indirect ELISA and western blotting. To develop a capture ELISA for chicken IFN-k, two sets of the best capture and detection mAb combinations were identified via pairing assays. To validate the antigen-capture assay, the production of native IFN-k was induced in chicken HD11 macrophages using polyinosinic: polycytidylic acid (poly I:C) and was detected using the newly developed capture ELISA. Furthermore, qRT-PCR was used to confirm the transcript-level expression of IFN-k in HD11 cells at 24 and 48 h. The neutralizing effects of anti-chIFN-k mAbs were evaluated based on their ability to block the induction of IFN-stimulated genes (ISGs) in DF-1 cells stimulated with recombinant chIFN-k 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-k. This novel antigen-capture ELISA should enhance the quality of fundamental and applied research involving IFN-k in the normal and diseased state.