Development and characterization of mouse monoclonal antibodies reactive with chicken CD80

Abstract This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 ( ch CD80). A recombinant plasmid containing a ch CD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant ch CD80/IgG4 protein. Chicken CD80 was purified from the ch CD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against ch CD80. Three mAbs with high binding specificity for recombinant ch CD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0 kDa proteins. Immunohistochemical analysis revealed ch CD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, ch CD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48 h increased ch CD80 expression. Finally, addition of ch CD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these ch CD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.