title: Development and characterization of mouse monoclonal antibodies reactive with chicken CD83.
abstract: This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.
authors.affiliation: Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA.
authors.forename: Sung Hyen
authors.initials: SH
authors.lastname: Lee
authors.forename: Hyun S
authors.initials: HS
authors.lastname: Lillehoj
authors.forename: Seung I
authors.initials: SI
authors.lastname: Jang
authors.forename: Kyung Woo
authors.initials: KW
authors.lastname: Lee
authors.forename: Cynthia
authors.initials: C
authors.lastname: Baldwin
authors.forename: Dannielle
authors.initials: D
authors.lastname: Tompkins
authors.forename: Bettina
authors.initials: B
authors.lastname: Wagner
authors.forename: Emilio
authors.initials: E
authors.lastname: Del Cacho
authors.forename: Erik P
authors.initials: EP
authors.lastname: Lillehoj
authors.forename: Yeong Ho
authors.initials: YH
authors.lastname: Hong
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology*; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D002645:Chickens / Q000276:immunology; D006224:Cricetinae; D003713:Dendritic Cells / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005434:Flow Cytometry / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D006825:Hybridomas / Q000276:immunology; D007136:Immunoglobulins / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice / Q000276:immunology; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000276:immunology; D000097802:CD83 Antigen
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D000097802:CD83 Antigen