Characterization of immunological properties of chicken chemokine CC motif ligand 5 using new monoclonal antibodies

Authors: LU, MINGMIN - US Department Of Agriculture (USDA); Panebra, Alfredo; KIM, KWOOHYUN - US Department Of Agriculture (USDA); Lillehoj, Hyun

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/19/2021
Publication Date: 1/23/2021
Citation: Lu, M., Panebra, A., Kim, K., Lillehoj, H.S. 2021. Characterization of immunological properties of chicken chemokine CC motif ligand 5 using new monoclonal antibodies. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2021.104023.
DOI: https://doi.org/10.1016/j.dci.2021.104023

Interpretive Summary: A lack of knowledge on the function of different components of chicken immune system hinders our application of immunological knowledge to develop antibiotic alternatives like vaccines and novel immunotherapeutics. Unlike the immunomodulatory roles of human and mouse CCL5, which have been thoroughly elucidated via commercially available assays, chCCL5 expression and its detection have been limited due to the absence of commercial detection kits. In this paper, ARS scientists describe new chicken immune reagents which detect the CC chemokine ligand 5, CCL5. This chemokine was first discovered through its ability to recruit leukocytes and is known to play a pivotal role in host defense against viral or parasitic infections. In this study, new anti-chCCL5 monoclonal antibodies (mAbs) were generated and their potential to detect native chicken CCL5 in various biological samples were verified. Because of their exquisite specificity toward chicken CCL5, these new tools will be valuable to study disease processes involving inflammation in fundamental and applied studies in poultry. Furthermore, potential commercial application of anti-chCCL5 mAbs and CCL5-specific antigen-capture detection ELISA for detecting native chCCL5 in biological samples in poultry is expected.

Technical Abstract: CCL5 (formerly RANTES) belongs to the CC (or ß) chemokine family and is associated with a plethora of inflammatory disorders and pathologic states. CCL5 is mainly produced and secreted by T cells, macrophages, epithelial cells, and fibroblasts and acts as a chemoattractant to recruit effector cells to the inflammation sites. Chicken CCL5 (chCCL5) protein is closely related to avian CCL5 orthologs but different from mammals orthologs, and its modulatory roles in the immune response are largely unknown. The present work was undertaken to characterize the immunological properties of chCCL5 using the new sets of anti-chCCL5 mouse monoclonal antibodies (mAbs). Eight different mAbs (6E11, 6H1, 8H11, 11G1, 11G11, 12H1, 13D1, and 13G3) were characterized for their specificity and binding ability toward chCCL5. Two (13G3 and 6E11) of them were selected to detect native chCCL5 in chCCL5-specific antigen-capture ELISA. Using 13G3 and 6E11 as capture and detection antibodies, respectively, the ELISA system detected serum chCCL5 secretions in Clostridium perfringens- and Eimeria-infected chickens. The intracellular expressions of chCCL5 in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 13G3 and 6E11 mAbs. Furthermore, 6E11, but not 13G3, neutralized chCCL5-induced chemotaxis in vitro using chicken PBMCs. These molecular characteristics of chCCL5 demonstrate the potential application of anti-chCCL5 mAbs and CCL5-specific antigen-capture detection ELISA for detecting native chCCL5 in biological samples. The availability of these new immunological tools will be valuable for fundamental and applied studies in avian species.