Biology Reference
In-Depth Information
Urine EPO concentrations by immunoassay
Attempts to detect abuse of erythropoietic proteins by urine immunoassay
have not been successful. Since rHuEPO and endogenous urinary EPO cannot
be distinguished by the available antibodies [61], a diagnostic test would
depend on urinary EPO concentrations that are far above the normal range. In
addition, <5% of the dose is excreted in urine [62] and urinary EPO concen-
trations are affected by pH, specific gravity, and exercise [63, 64]. Small
increases in urinary EPO were detected by a immunoradiometric assay after
large doses (200 U/kg every other day for 10 days) [61].
Direct tests for doping with erythropoietic proteins
The isoelectric focusing test for urinary rHuEPO
A method for detecting rHuEPO in urine by electrophoresis was first described
in 1995 [65]. Although this test had practical limitations, it demonstrated con-
clusively that the isoform pattern of urinary endogenous EPO differs from the
pattern of urinary rHuEPO, and it was the first successful attempt to develop a
direct test for urinary rHuEPO. Similarly in 2002, Skibeli et al. [66] isolated
EPO from human serum and showed, using gel electrophoresis, that endoge-
nous and recombinant EPO differed.
A significant improvement in practical detection occurred in when Lasne
and de Ceaurriz [67] described a method for detecting rHuEPO in urine based
on isoelectric focusing with immunoblotting plus one novel and critical step:
a second blot (“double-blotting”) [68]. After the isoforms of rHuEPO are sep-
arated by isoelectric focusing, the first blot is performed, then the membrane
containing the transferred proteins is incubated with anti-EPO antibody. The
second blot transfers only the anti-EPO antibodies to a second membrane, and
the second membrane is incubated with a second antibody directed against the
first antibody. This step markedly reduces non-specific binding and yields
clear isoform patterns. After the second antibody is incubated with strepta-
vidin-horseradish peroxidase and substrate, the emitted chemiluminescence is
captured to produce an image of the gel [69].
This method shows that endogenous EPO is highly heterogeneous and is
composed of a number of isoforms. Most of these isoforms are not present in
the recombinant hormones. After administration of rHuEPO or darbepoetin
alfa (a new erythropoeisis-stimulating protein with a longer half-life than
rHuEPO), a striking change in urinary EPO pattern is observed with the
appearance of new isoforms corresponding to the excretion of the injected
drugs and,after sufficient doses, the disappearance of the endogenous iso-
forms. By analyzing the isoelectric pattern it is possible to determine if the
excreted EPO is of natural or recombinant origin. Figure 1 is an electrophero-
gram showing the band pattern of darbepoetin alfa in a urine obtained from a
patient who was treated with darbepoetin alfa.
