Development of a sandwich ELISA for the detection of chicken colony stimulating factor 1a

Authors: Panebra, Alfredo; Lillehoj, Hyun

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/15/2022
Publication Date: 4/26/2022
Citation: Panebra, A., Lillehoj, H.S. 2022. Development of a sandwich ELISA for the detection of chicken colony stimulating factor 1a. Poultry Science. https://doi.org/10.1016/j.psj.2022.101924.
DOI: https://doi.org/10.1016/j.psj.2022.101924

Interpretive Summary: There is a timely need to identify and research the function of various immune cells and the immune factors that they secrete to develop effective immunotherapeutic strategies to reduce the use of antibiotics to treat infectious diseases in poultry. In this report, ARS scientists describe a new chicken factor called colony stimulating factor-1, CSF-1, which is secreted by macrophages. Chicken CSF-1 stimulates the survival, proliferation, and differentiation of the mononuclear phagocytes and is involved in bone metabolism, fertility, inflammatory processes, and homeostasis. To better understand the function of CSF-1 in chicken disease processes, we developed five mouse monoclonal antibodies (mAbs) that specifically detect chicken CSF-1 protein and used them to develop a sensitive immunoassay to measure the production of native chicken CSF factor during enteric diseases. The results showed that this new immunoassay for chicken CSF-1 can detect the production of CSF-1 factor in serum samples which were obtained from commercial broiler chickens infected with coccidiosis, a parasitic disease affecting all poultry production. Therefore, new mAb-based sandwich assay will enhance the knowledge discovery of CSF-1 factor in normal and disease states.

Technical Abstract: Macrophage colony stimulating factor-1 (M-CSF-1 or CSF-1) is a hematopoietic growth factor that stimulates the survival, proliferation, and differentiation of the mononuclear phagocyte lineage and is involved in bone metabolism, fertility, pregnancy, inflammatory processes, and homeostasis. CSF-1-activated macrophages display unique features, such as distinguishable cell surface antigens, enhanced Fc-'-receptor-mediated phagocytosis, intensified reactive oxygen species activity, enhanced proliferation, and enhanced chemotaxis. Five mouse monoclonal antibodies (mAbs) for the detection of chicken CSF-1 were developed and characterized using western blot, indirect ELISA, and in vitro functional assays. One of the anti-chCSF-1 mAbs, 8A12, showed neutralization of macrophage proliferation and NO release, whereas mAb 1G4 inhibited the phagocytosis of fluorescent-labeled bacteria by HD11 cells in vitro. For the quantitative assessment of native chCSF-1 in biological samples from chickens, a sensitive sandwich ELISA was developed using the best capture and detection pair of mAbs that were selected from five anti-chCSF-1 mAbs. Chickens challenged with Eimeria acervulina, E. maxima, and E. tenella presented a steady increase in the circulating levels of CSF-1, starting from day 1 and steadily increasing until day 7 post-challenge, reaching their peak at day 10 post-challenge. The CSF-1 synthesis kinetic patterns induced by the three Eimeria species tested were quite similar, even though these species were reported to be phenotypically and immunologically different. Therefore, an mAb-based sandwich ELISA will be a valuable tool for the detection of CSF-1 production during infections, and these new anti-chCSF-1 mAbs will facilitate the fundamental and applied research related to CSF-1 function in inflammation and homeostasis in chickens.