An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control.

Recombinant erythropoietins (rEPOs) are still among the substances endurance athletes use for doping. Detection methods are based on an electrophoretic separation of the proteins followed by a western blot and immunodetection with specific anti-EPO antibodies. In addition to IEF-PAGE, the SDS-PAGE method has been used to differentiate endogenous EPO from rEPOs by their molecular weight (MW).

Improved detection methods significantly increase the detection window for EPO microdoses.

To reproduce a potential doping scenario, a 2 week administration of recombinant erythropoietin (rEPO) microdoses alone or in combination with growth hormone (GH) microdoses (three times a week) was performed on healthy and athletic male subjects. The aim of this study was to evaluate the identification capability of rEPO in samples obtained during and post treatment. Detection was tested in urine and blood using the antidoping techniques for rEPO detection (iso-electric focusing (IEF)-, sodium-dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and for some urine samples the sarcosyl (SAR)-PAGE method) with some improvements: for blood samples, instead of a simple concentration step, immuno-extraction of EPO was performed for all urines to limit protein contamination that can affect migration.

Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods.

The combination of growth hormone (GH) and recombinant erythropoietin (rEPO) is thought to be used particularly in endurance sports. Our objective was to reproduce a 2-week administration of rEPO microdoses alone or in combination with GH microdoses (three times a week) on healthy and athletic male subjects and to evaluate if GH had any additional effects compared to EPO treatment alone. The effects of the treatments on hematological parameters and VO2max were studied as well as the detection of GH in serum.

Cave digitabula!

We report here observations clearly demonstrating that gloves can have a deleterious impact on an IEF experiment. These observations were made during the practice of analyses for anti-doping control of erythropoietin. We show that the wearing of nitrile gloves to apply the catholyte strip onto the IEF gel may be responsible for dramatic distortions in the pattern of this hormone.

Isoelectric profiles of human erythropoietin are different in serum and urine.

By adding a step of immunoaffinity to the method we had previously developed for analysing erythropoietin (EPO) in urine, we were able to study the isoelectric profiles of this hormone in human serum samples. This method was sensitive enough to investigate samples presenting physiological levels of this hormone. Comparison with the corresponding profiles in urine showed that natural EPO was systematically more acidic in urine.

Detection of recombinant epoetin and darbepoetin alpha after subcutaneous administration in the horse.

A direct detection method for anti-doping control of recombinant human erythropoietin (rHuEPO) abuse in racehorses is proposed. This method involves screening of plasma (or serum) by an enzyme-linked immunosorbent assay specific for human EPO and confirmation in urine samples by characterization of the urinary EPO isoelectric profile. This method was tested on horses that were administered epoetin alpha (rHuEPO) and the hyper-glycosylated form of this drug (darbepoetin alpha).