Erythropoietin (EPO) - Detection Method

The current method for detecting EPO approved by the World Anti-Doping Agency (WADA) involves the direct detection of EPO in urine or blood.

Genetically engineered EPO preparations (recombinant EPO) differ from EPO (endogenous EPO), which is synthesized in the human body, due to a high degree of heterogeneity in the sugar chains. Differences are known to exist in the branching patterns of the chains, the number of terminal sialic acid groups, and the number of sulfate groups.

In addition, there are recombinant EPO preparations that exhibit differences in amino acid sequence (e.g., darbepoetin) or are additionally pegylated (conjugated with polyethylene glycol) (e.g., CERA). These differences can be analytically exploited using electrophoretic separation systems and utilized for differentiation in the detection of doping.

For electrophoretic methods, either the IEF method (isoelectric focusing), first introduced by Francoise Lasne for doping analysis, or the SDS-PAGE or SAR-PAGE method can be used. The latter two methods separate the different forms of EPO based on molecular size.

SDS-PAGE is the abbreviation for sodium dodecyl sulfate polyacrylamide gel electrophoresis;

SAR-PAGE is the abbreviation for sarcosyl polyacrylamide gel electrophoresis. PDF

download: Urine IEF method

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Interpretation of the Results

To interpret the results, the raw data file is processed using a certified analysis program (GASEPO), during which all bands are contrast-optimized. Using the Dynepo reference standards, a line (blue) is superimposed across the entire gel. All negative samples show signals below this line, while recombinant EPO preparations exhibit a signal above the blue line. In the positive doping control sample (Lane 19), a very broad signal is detected both above and below the blue reference line. This indicates that, in addition to recombinant EPO, traces of endogenous (endogenous) EPO are also present in the urine sample.