Proteome Systems develops EPO test for drug cheat athletes

Sydney company Proteome Systems has developed a new test to unmask endurance athletes who try to gain an illegal aerobic edge with the red blood-cell booster erythropoietin (EPO).

The World Anti-Doping Agency (WADA) is funding a project by Proteome Systems last year to develop a simpler, more accurate test for EPO.

The Sydney company's automated 2D gel electrophoresis technology can readily distinguish between naturally occurring and recombinant variants of protein-based drugs.

WADA was said to have approached Proteome Systems to develop a simpler, more robust test to detect EPO in the urine of cheating athletes.

The new test, developed by Dr Nicolle Packer's research group at Proteome Systems, is likely to replace a test developed by French researchers for the Sydney Olympics four years ago.

While the new test will not be deployed in time for the Athens Olympics, it will be in use before the Beijing Olympics in 2008. It will make it virtually impossible for athletes using recombinant (genetically engineered) EPO to escape detection.

The EPO test zeroes in on subtle differences in electrical charge between naturally occurring forms, or isoforms, of the proteins, and recombinant forms produced in bioreactors. The charge differences arise in different patterns of glycosylation -- the attachment of sugar residues at various points along the protein amino acid chain.

Packer said that where native EPO exists in half a dozen isoforms in human blood, all differing in charge patterns, recombinant EPOs exhibit a single, distinctive pattern.

The current French test for EPO uses one-dimensional gel electrophoresis, followed by two Western blots, which use EPO-specific antibodies to detect the protein and confirm it is actually EPO.

Packer says the 1D gel electrophoresis techniques leave EPO overlaid by other proteins of similar charge. The first Western blot is used to segregate the proteins, while the second identifies it as EPO. She said the Proteome Systems 2D gel electrophoresis system sorts the proteins on the basis of both charge and mass, and requires only one Western blot.

Because it is fully automated, it provides consistent, repeatable results. Proteome Systems' special wrinkle is to use a reference glycoprotein of similar molecular weight and charge to EPO, which co-migrates with EPO, flagging its position so it can be robotically excised for the Western blot test.

Packer said the technology was readily applicable to other illegal performance-enhancing proteins, including the muscle-growth agents human growth hormone (hGH) and insulin-like growth factor-1 (IGF1).

"HGH is an interesting protein because it's not glycosylated," she said. "But it exists in three different forms in the body, whereas the recombinant version has a single isoform, so we can separate on that basis."

New, long-acting versions of EPO developed by Amgen and Roche to treat anaemia in kidney disease patients will be readily detected by the Proteome Systems test if athletes illegally use them.

Packer said the Amgen product, already in clinical use, has been modified to create extra glycosylation sites. Roche's experimental drug, now in Phase III clinical trials, employs a polymer, integrated into the amino acid chain, to extend its half life.

Packer said the Australian Government Analytical Laboratory, in Sydnet's northern suburbs, is currently validating the new EPO test before it goes to WADA. It will then be up to the International Olympic Committee's own drug-testing agency to introduce it.

Packer believes the test will save lives, especially among endurance cyclists in major events like the Tour de France. "It's amazing what these guys will do," she said. "There have been cases where cyclists have dropped dead because their blood is so thick their hearts can no longer pump it around the body. They know there is a chance of that happening when they use EPO, but they use it anyway."

Packer said some athletes, including cyclists, were still employing the dangerous practice of training at altitude, then taking their own blood and concentrating it, then infusing it back into their bodies to improve their endurance. Because the athlete's own EPO was being used, no test for EPO could detect it.