title: Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25).
abstract: This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.
authors.affiliation: Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, US 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: 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: Mark
authors.initials: M
authors.lastname: Parcells
authors.forename: Emilio
authors.initials: E
authors.lastname: Del Cacho
authors.forename: Yeong Ho
authors.initials: YH
authors.lastname: Hong
authors.forename: Wongi
authors.initials: W
authors.lastname: Min
authors.forename: Erik P
authors.initials: EP
authors.lastname: Lillehoj
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D015153:Blotting, Western / Q000662:veterinary; D002060:Bursa of Fabricius / Q000276:immunology; D016466:CHO Cells; D002645:Chickens / Q000276:immunology; D006224:Cricetinae; D004386:Duodenum / Q000276:immunology; D005434:Flow Cytometry / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D006825:Hybridomas / Q000276:immunology; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D053645:Interleukin-2 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins