title: Production of monoclonal antibodies against human erythropoietin and their use in the purification of human urinary erythropoietin.
abstract: Several murine monoclonal antibodies (MAbs) specific for human erythropoietin (HuEpo) were produced by hybridomas obtained from the fusion of murine myeloma cells, P3X63-Ag.8-653, with the splenocytes of mice immunized with recombinant human Epo (rHuEpo). Based on epitope analysis by a competitive binding assay, these MAbs could be classified into at least three groups: (1) 1E10, (2) 1H7, (3) 2D6, 3D6 and 3D8. In a sandwich enzyme-linked immunosorbent assay (ELISA), using these MAbs as the solid-phase antibodies, MAb-bound HuEpo was detected with rabbit anti-HuEpo sera. Some combinations of two different classes of MAbs, such as 1H7 and 3D8, were found to capture much more HuEpo than each MAb used individually. Urinary HuEpo (U-HuEpo) was highly purified from the urine of patients with severe aplastic anemia with about 50% final recovery using an immunoaffinity column on which a mixture of 1H7 and 3D8 was immobilized. The purified U-HuEpo had a specific activity of 77,340 U/mg in a radioimmunoassay (RIA) and of 76,673 U/mg using an in vivo bioassay.
authors: Pharmaceutical Laboratory, Kirin Brewery Co., Ltd., Gunma, Japan.
authors: H
authors: H
authors: Miyazaki
authors: H
authors: H
authors: Kozutsumi
authors: T
authors: T
authors: Kato
authors: S
authors: S
authors: Hoshi
authors: S
authors: S
authors: Tamura
authors: M
authors: M
authors: Kubota
authors: T
authors: T
authors: Suzuki
mesh_terms: D000741:Anemia, Aplastic / Q000652:urine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology; D004921:Erythropoietin / Q000276:immunology* / Q000302:isolation & purification / Q000652:urine; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D004921:Erythropoietin