title: Fc-fragment removal allows the EPO-Fc fusion protein to be detected in blood samples by IEF-PAGE.
abstract: EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.
authors.affiliation: Moscow Antidoping Centre, Russian Federation, Moscow, 105005, Elizavetinsky per., 10/1, Russia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Postnikov
authors.affiliation: Moscow Antidoping Centre, Russian Federation, Moscow, 105005, Elizavetinsky per., 10/1, Russia.
authors.forename: Grigory
authors.initials: G
authors.lastname: Krotov
authors.affiliation: Moscow Antidoping Centre, Russian Federation, Moscow, 105005, Elizavetinsky per., 10/1, Russia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Mesonzhnik
authors.affiliation: Moscow State university of fine chemical technologies, Russian Federation, Moscow, 119571, Vernadsky pr., 86, Russia.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Efimova
authors.affiliation: Moscow Antidoping Centre, Russian Federation, Moscow, 105005, Elizavetinsky per., 10/1, Russia.
authors.forename: Grigory
authors.initials: G
authors.lastname: Rodchenkov
mesh_terms: D015153:Blotting, Western; D002138:Calibration; D002853:Chromatography, Liquid; D004300:Doping in Sports; D004591:Electrophoresis, Polyacrylamide Gel / Q000592:standards; D004921:Erythropoietin / Q000097:blood*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000097:blood*; D007525:Isoelectric Focusing / Q000592:standards; D007526:Isoelectric Point; D008163:Luminescent Measurements; D010447:Peptide Hydrolases / Q000378:metabolism; D058573:Performance-Enhancing Substances / Q000097:blood*; D011237:Predictive Value of Tests; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry
chemical_list: D007141:Immunoglobulin Fc Fragments; D058573:Performance-Enhancing Substances; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D010447:Peptide Hydrolases
keywords: EPO-Fc fusion protein; EPO-hinge fragment; IEF-PAGE; IdeS protease; doping control