| Autor: |
Postnikov, Pavel, Krotov, Grigory, Mesonzhnik, Natalia, Efimova, Yulia, Rodchenkov, Grigory |
| Zdroj: |
Drug Testing & Analysis; Nov/Dec2015, Vol. 7 Issue 11/12, p999-1008, 10p |
| Abstrakt: |
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. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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