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Name: Scott W.
Status: Educator
Age: 30s
Location: N/A
Country: N/A
Date: September 2002


Question:
I know they use electrophoresis to test for r-Erythropoetin, but how can they distinguish between what natural and what's recombinant?



Replies:
Dear Scott:

According to the following interesting article on "Blood Testing for Professional Cyclists" by Dr. David T. Martin of the Australian Inst. of Sport, on the Sportscience Peer-Reviewed web site for Sport Research ( http://www.sportsci.org/news/news9703/AISblood.html ), "the electrophoretic technique is based on the observation that endogenous EPO generally has a greater negative charge than rhEPO." ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_u ids=8587495&dopt=Abstract ). Commercial rhEPO is produced from Chinese hamster ovary (CHO) cells. EPO is modified by glycosylation within the cell after the protein itself is synthesized, and CHO cells generate a slightly different pattern of glycosylation than the endogenous human form, which probably accounts for the observed charge differential.

Interestingly, the new, longer lasting version of rhEPO, known as Aranesp, is artificially hyperglycosylated. I have heard that it is more similar to the native form of EPO, although I think it is still produced in CHO cells, and that it is more difficult to distinguish from the endogenous form, probably because the hyperglycosylation increases its negative surface charge. There is more info. about Aranesp in this PDF slide show from Dr. Graham Molineaux of Amgen, although I had a lot of trouble with it freezing my browser ( http://www.aei.org/past_event/molineux.pdf ).

Thanks a lot for the very interesting question,

Jeff Buzby, Ph.D.
Children's Hospital of Orange County



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