The detection method of rHuEPO levels in blood and
urine samples is based on direct detection of their presence, and on indirect
detection by haematological parameter based measurements. The laboratory
statistical analysis from WADA have announce that since 2003 the detection of
rHuEPO misuse in athletes has gone down by 51 to 41 in 2015 (WADA et al. 2015).
The standard direct protocol currently used to detect rHuEPO doping in
competitive sports, isoelectric focusing (IEF) and double blotting. These
direct methods aren’t very practical, as the short biological half-life of
rHuEPO makes it only detectable in urine for 3-7 days (Lamon S. et al., 2010).
IEF is a separation technique, exploiting the difference in CHO composition
between the two EPO. The detection is done by showing that there are fewer isoforms
of rHuEPO than urinary eEPO (Reichel et al., 2011). Consequently, the overall
charge of rHuEPO is less negative than that of eEPO, the isoform of eEPO
migrate in a more acidic IEF gel (e.g. pH ranging between 2-6) than rHuEPO in
more basic are. The double-blotting is done using monoclonal AE75 antibody,
before the isoforms are visualized by chemiluminescence (Pottgiesser and
Schumacher et al., 2013). Eventually the resulting analysis of IEF pattern of
the EPO molecules presented in the urine samples. The indirect detection method
of detecting rHuEPO gene expression difference is by analysis glycopeptide
fragments. The study focused on using magnetic beads combined with anti-rHuEPO
antibody to extract the EPO’s from a urine sample and then digested the glycopeptide
into fragments with trypsin enzyme, with methodology of Nano-liquid
chromatography-tandem mass spectrometry. There was this one unique peptide
known as T6 corresponding to amino acid sequence VNFYAWK. The MS
detection was able to identify that the peptide T6 was unique to the EPO’s
(e.g. rHuEPO/DPO/PEG-EPO) and could be differentiated from the corresponding T6
in endogenous EPO (VNFYSWK) based on one amino acid difference in the
sequence (Nola H. et al., 2010). There is indirect detection of other gene
expression pattern, that focus on alteration shown in genes such as
haemoglobin-? (HBB), ferritin-light chain (FTL) and ornithine decarboxylase
antizyme (OAZ)(R).

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