Enzyme-linked immunosorbent assay detects a potential soluble form of the erythropoietin receptor in human plasma.
The erythropoietin receptor (EpoR) is a type I transmembrane protein that is a member of the family of hemopoietin receptors. Several members of this family have soluble receptor forms that are secreted by the cells rather than expressed on the cell surface. An alternatively spliced EpoR transcript has been described in human erythroid precursors that, if translated, would produce a truncated, soluble EpoR lacking the transmembrane domain. To determine if the human EpoR is expressed in a soluble form, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for the EpoR, and we analyzed human serum and plasma. Sheep were immunized with a fusion protein (EREx) consisting of glutathione-S-transferase (GST) and the human EpoR extracellular domain. The sheep antiserum was affinity-purified on immobilized EREx, and then used in a two-stage antigen capture ELISA. The plasma from 20 normal subjects was studied with this assay. There was a wide variability in the levels of soluble EpoR in these subjects (range, <10-2,200 ng/ml). An average value of 550 +/- 735 ng/ml for soluble EpoR was obtained in these normals. Protein A adsorption of the test plasma prior to the assay had no effect on the values obtained. Assay of serum from the same normal subjects showed an average decrease of 88% in soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR level. This assay may have utility in the further elucidation of erythropoietin physiology.
Harris
K W
KW
Department of Medicine, University of Texas Health Science Center at San Antonio, USA.
Winkelmann
J C
JC
D000328:Adult; D000818:Animals; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006400:Hematocrit; D006801:Humans; D008297:Male; D008970:Molecular Weight; D010949:Plasma; D012326:RNA Splicing; D017467:Receptors, Erythropoietin / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012756:Sheep; D012995:Solubility
D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins