title: Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.
abstract: The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc-part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO-Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance-enhancing agents, EPO-Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO-Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO-Fc from human serum via high capacity protein A beads and subsequently detects EPO-Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO-Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS-PAGE, SAR-PAGE) and Western double-blotting with chemiluminescence detection - a method already established in routine EPO anti-doping control. The latter strategy allows the detection of EPO-Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol.
authors: Doping Control Laboratory, AIT Seibersdorf Laboratories, A-2444 Seibersdorf, Austria. christian.reichel@seibersdorf-laboratories.at
authors: Christian
authors: C
authors: Reichel
authors: Mario
authors: M
authors: Thevis
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D007525:Isoelectric Focusing; D057230:Limit of Detection; D013058:Mass Spectrometry; D011993:Recombinant Fusion Proteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: C554270:EPO protein, human; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin