Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4

Authors: LU, MINGMIN - US Department Of Agriculture (USDA); KIM, WOO H - US Department Of Agriculture (USDA); Lillehoj, Hyun; Li, Charles

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/7/2020
Publication Date: 9/1/2020
Citation: Lu, M., Kim, W., Lillehoj, H.S., Li, C.Z. 2020. Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4. Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.vetimm.2020.110091.
DOI: https://doi.org/10.1016/j.vetimm.2020.110091

Interpretive Summary: Fundamental information on how host innate immune cells in chickens get activated by external stimuli is poorly understood. To better understand poultry innate immunity to improve host disease resistance to environmental infectious agents, we need better immunological tools to identify these cells. ARS scientists isolated a chicken gene which encodes macrophage inflammatory protein (MIP)-1 which is a small, and highly conserved protein. MIP-1ß, also known as chemokine ligand, CCL4, is one of the four MIP-1 proteins that was identified 20 years ago with its physiological roles still being investigated. In mammals. CCL4 is produced by a wide range of cells and is induced by various pro-inflammatory stimuli including lipopolysaccharide (LPS), viral infections, and cytokines. Mammalian CCL4 acts as a chemoattractant to a variety of immune cells including T cells, monocytes and natural killer cells. Because of its crucial involvement in various biological functions of this protein, basic studies on this CCL4 in chickens will provide crucial information that will help to develop a better vaccine against important infectious agents of poultry. The results of this study proved important physiological function of chicken CCL4 and more importantly, several new mouse monoclonal antibodies have been developed and a novel assay to measure CCL4 has been developed for the first time. These immune tools will enhance our ability to study biological function of CCL4 to enhance basic and applied poultry research in normal and disease states.

Technical Abstract: Chemokine (C-C motif) ligand (CCL)4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated > 20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of > 400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture ELISA, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.