A hybridoma cell line producing the monoclonal antibody against erythropoietin 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 with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources. Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons.

Topics:
epitope mapping, erythropoietin, monoclonal antibodies, receptor protein-tyrosine kinases, erythropoietin receptors, antigens, western blotting, enzyme immunoassay, peptide library, cell lines
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Copyright © 1996 by The American Society of Hematology
1996
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