Beullens, M., Delanghe, J. R., & Bollen, M. (2006). False-positive detection of recombinant human erythropoietin in urine following strenuous physical exercise. Blood, prepublished online February 21, 2006 [https://www.bloodjournal.org/cgi/content/abstract/2006-01-0028v1]

Abstract

Erythropoietin (EPO) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human EPO (rhEPO) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEPO in urine. The adopted test is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and recombinant human EPO. We show here that this widely-used test can occasionally lead to the false-positive detection of rhEPO (EPOetin-) in post-exercise, protein-rich urine, probably because the adopted monoclonal anti-EPO antibodies are not monospecific.

Implications. The following comments were extracted from the Doping Journal [https://dopingjournal.org/mydopingjindex.html].

The study published in the major international scientific journal on blood science and medicine suggests International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) testing for Erythropoietin (EPO) is based on faked science.

Erythropoietin (EPO) is a glycoprotein hormone that is mainly produced by the kidney. It boosts the production of red blood cells by promoting the proliferation, differentiation and survival of progenitor cells of the erythroid lineage. Recombinant human EPO (rhEPO) is widely used for the treatment of various forms of anaemia. Since rhEPO increases the body's maximum oxygen consumption capacity and endurance by increasing red cell mass, it has also been embraced as an aid in endurance sports. However, this use of EPO was prohibited by IOC and WADA , which has led to the screening of athletes for rhEPO abuse.

Endogenous and recombinant human forms of EPO have different patterns of molecule modification called glycosylation. A tiny difference between natural EPO and its biotechnological and structural analogs (such as DarbEPOetin) leads to a molecule electric charge difference that has been exploited to distinguish endogenous and recombinant isoforms by a so called isoelectric focusing technique.

The WADA-adopted urine test for EPO is based on a combination of isoelectric focusing on a gel (a semi-analytical separation of proteins according to the molecules' net electric charge), the transfer of proteins from gel to a special paper, and biomolecules detection by a double immunoblotting. This is not a well established procedure to immunologically detect proteins of interest with an antibody color reaction. It iscomplicated and operator-dependent.

In the Blood journal study, a research group led by Associate Professor Monique Beullens and Professor Mathieu Bollen (of the Department of Molecular Cell Biology, Faculty of Medicine, Catholic University of Leuven, Leuven, Belgium) and their colleague Dr. Joris R Delanghe (of the Department of Clinical Chemistry, University Hospital, Gent, Belgium), showed the widely-used IOC and WADA EPO test can lead to the false-positive detection of rhEPO in post-exercise, protein-rich urine of endurance sports athletes.

As a result of a disputed case of alleged rhEPO-abuse by an endurance athlete with post-exercise proteinuria, European scientists wondered whether the test for rhEPO could lead to false-positive results, perhaps as a result of cross-reactivity of the EPO-antibodies with other proteins of urine that were unrelated to EPO.

The straightforward experimental protocol of the reported study leaves little doubt that the major urinary protein that the WADA test visualizes with the EPO antibodies is not EPO.

The article by Belgian scientists therefore challenges WADA claim that "the detection method for EPO is valid and reliable". Other WADA statements such as the method for EPO detection "has undergone an extensive scientific validation" and "it is a well-established procedure widely accepted by the scientific community, as demonstrated by publication in a number of international scientific journals" are similarly false.

Contrary to the WADA claims, the Doping Journal analysis of citation impact of earlier publications on EPO testing in urine indicates the IOC/WADA method for EPO testing is not scientifically popular or well-established. An in-depth analysis of the articles behind the IOC's urine test for EPO showed earlier publications missed critical control experiments and were not designed to exclude non-specific false-positive misidentification of other non-EPO urine components.

Therefore, "The 2006 Prohibited List" section S2 on "Hormones and Related Substances" of The World Anti-Doping Code (stating with regard to EPO that "unless the athlete can demonstrate that the [EPO] concentration was due to a physiological or pathological condition" [the urine sample] is deemed to contain the prohibited substance EPO). This blatant error provides grounds for blood doping corruption by the IOC and WADA, it abuses athletes, and it contradicts the Olympic "play true" principle.

The study published in an advanced online publication of the journal Blood, suggested that WADA data on blood doping are invalid, and that innocent athletes could lose Olympic medals because of IOC and WADA misconduct. The latest possible IOC false detection of rhEPO took place at Salt Lake City during Winter Olympic Games 2002, when two Russian cross-country skiers (Larissa Lazutina and Olga Danilova) and one Spanish athlete (Johann Muehlegg) were stripped off their medals after testing positive for the EPO analog Darbepoietin. The present Blood study published during the XX Olympic Games 2006 in Turin, Italy, indicates Olympic champions could wrongly suffer IOC sanctions.

Other aspects of the misconduct by WADA are analyzed in the leading article of the just published first issue of the newly established International Journal of Sports Science & Coaching [https://www.multi-science.co.uk/sports-science&coaching.htm].

[Blood (https://www.bloodjournal.org/?dj ) is the journal of the American Society of Hematology, the major peer reviewed international journal on science and medicine of blood with very high latest impact factor of ~10.

Doping Journal (https://www.dopingjournal.org/) is an independent non-profit international peer-reviewed scholar publication on every aspect of doping science. It is a so called Open Access (OA) publication available free of charge for everyone with a computer and an Internet connection. It is indexed by the National Library of Medicine USA, Directory of Open Access Journals (DOAJ), and a great number of academic and public libraries worldwide.]

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