ANTIERYTHROPOIETIN RECEPTOR MONOCLONAL-ANTIBODY - EPITOPE MAPPING, QUANTIFICATION OF THE SOLUBLE RECEPTOR, AND DETECTION OF THE SOLUBILIZEDTRANSMEMBRANE RECEPTOR AND THE RECEPTOR-EXPRESSING CELLS

A hybridoma cell line producing the monoclonal antibody against erythr opoietin receptor (EpoR) was established using the soluble ectodomain of mouse erythropoietin receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR, Epitope mapping wit h peptide library revealed that 1G3 recognized the amino terminal regi on including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rat s, and humans, and therefore 1G3 bound to EpoR from all of these sourc es, Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoas say, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immun ochemical staining. We confirmed the presence of EpoR in a neuronal ce ll line and PC12 cells, and EpoR was expressed in primary cultured hip pocampal neurons. (C) 1996 by The American Society of Hematology.