title: Identification of a sensitive anti-erythropoietin receptor monoclonal antibody allows detection of low levels of EpoR in cells.
abstract: Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on non-erythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the EpoR protein on intact cells by flow cytometry and by western blot analysis with cell lysates. Both methods were optimized and using them, EpoR protein was detected by western immunoblotting with lysates from fewer than 200 EpoR positive control cells and the positive signals were proportional to EpoR protein expression level with a minimal signal in EpoR negative cells. The proteins detected by western blot analysis using A82 included full-length EpoR ( approximately 59kDa) as well as smaller EpoR fragments derived from the EPOR gene. These results indicate that A82 can be used to examine low level EpoR expression in cells by western and flow cytometry allowing an improved understanding of EpoR expression and metabolism.
authors.affiliation: Amgen Inc, One Amgen Center, Thousand Oaks, CA 91320, United States. selliott@amgen.com
authors.forename: Steve
authors.initials: S
authors.lastname: Elliott
authors.forename: Leigh
authors.initials: L
authors.lastname: Busse
authors.forename: Ian
authors.initials: I
authors.lastname: McCaffery
authors.forename: John
authors.initials: J
authors.lastname: Rossi
authors.forename: Angus
authors.initials: A
authors.lastname: Sinclair
authors.forename: Chris
authors.initials: C
authors.lastname: Spahr
authors.forename: Susan
authors.initials: S
authors.lastname: Swift
authors.forename: C Glenn
authors.initials: CG
authors.lastname: Begley
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002457:Cell Extracts; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007114:Immunization; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D017467:Receptors, Erythropoietin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D010446:Peptide Fragments; D017467:Receptors, Erythropoietin