Production of monoclonal antibodies against human erythropoietin and their use in the purification of human urinary erythropoietin

Miyazaki Hiroshi Miyazaki*, Kozutsumi Hiroyuki Kozutsumi, Kato Takashi Kato, Hoshi Sakuo Hoshi, Tamura Setsuko Tamura, Kubota Mamoru Kubota, Suzuki Takamoto Suzuki

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)261-267
Number of pages7
JournalJournal of Immunological Methods
Volume113
Issue number2
DOIs
Publication statusPublished - 1988 Oct 26
Externally publishedYes

Keywords

  • Erythropoietin
  • Immunoaffinity chromatography
  • Monoclonal antibody

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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