Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing

Drug Test Anal. 2012 Nov;4(11):818-29. doi: 10.1002/dta.1381. Epub 2012 Jul 5.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Erythropoietin / blood*
  • Erythropoietin / chemistry
  • Erythropoietin / isolation & purification
  • Humans
  • Immunoglobulin Fc Fragments / blood*
  • Immunoglobulin Fc Fragments / chemistry
  • Immunoglobulin Fc Fragments / isolation & purification
  • Isoelectric Focusing
  • Limit of Detection
  • Mass Spectrometry
  • Recombinant Fusion Proteins / blood*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Substance Abuse Detection / methods*

Substances

  • EPO protein, human
  • Immunoglobulin Fc Fragments
  • Recombinant Fusion Proteins
  • Erythropoietin