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Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium
Published on: May 18, 2021
Development and characterisation of monoclonal antibodies reactive with porcine CSF1R (CD115)
L Moffat1, L Rothwell1, C Garcia-Morales1
1The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK.
Insights
Researchers developed three monoclonal antibodies targeting porcine colony-stimulating factor 1 receptor (CSF1R). These antibodies are valuable tools for studying pig monocyte and macrophage biology without blocking CSF1 signaling.
Area of Science:
- Immunology
- Cell Biology
- Veterinary Science
Background:
- Colony-stimulating factor 1 (CSF1) and interleukin-34 (IL-34) regulate mononuclear phagocyte system cells by binding to the CSF1 receptor (CSF1R).
- Understanding CSF1R expression and function in pigs is crucial for advancing monocyte and macrophage research.
Purpose of the Study:
- To produce and characterize novel monoclonal antibodies against the porcine CSF1R.
- To evaluate the utility of these antibodies as research tools in pig immunology.
Main Methods:
- Production of a porcine CSF1R-Ig+pFUSE Fc fusion protein as an immunogen.
- Generation of three monoclonal antibodies (ROS8G11, ROS3A5, ROS3B10) against porcine CSF1R.
- Validation of antibody specificity and function using ELISA, Western blot, flow cytometry, and immunocytochemistry.
Main Results:
- Specific binding of the generated monoclonal antibodies to porcine CSF1R was confirmed.
- The antibodies did not inhibit CSF1-mediated signaling.
- Porcine CSF1R was found on CD14-positive monocytes and lung macrophages, with expression levels changing during bone marrow-derived macrophage (BMDM) differentiation.
Conclusions:
- The developed monoclonal antibodies are specific reagents for detecting porcine CSF1R.
- These antibodies facilitate the investigation of monocyte and macrophage biology and CSF1R dynamics in pigs.
Abstract:
Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system. CSF1, alongside a second ligand, interleukin-34 (IL-34), acts by binding to a cell surface receptor (CSF1R). We previously cloned and expressed pig CSF1 and IL-34. Here we produced a pig CSF1R-Ig+pFUSE Fc fusion protein and used it as an immunogen to produce three monoclonal antibodies (ROS8G11, ROS3A5 and ROS3B10) targeted against porcine CSF1R. Specific binding of each monoclonal antibody was confirmed by ELISA, Western blot, flow cytometry and immunocytochemistry. The antibodies did not block CSF1 signalling. The surface expression of CSF1R in pig peripheral blood was restricted to CD14-positive monocytes and was also detected on lung macrophages. These antibodies provided an opportunity to investigate the increase of available CSF1R during pig BMDM differentiation. The new monoclonal antibodies provide useful reagents to support the study of monocyte and macrophage biology in the pig.
