Detection of continuous erythropoietin receptor activator in blood and urine in anti-doping control

Abstract

by lymphocytes and it is expected that its levels steadily increase together with the progressive expansion of the leukemic clone suggesting a close correlation between stage (which measures tumor burden) and β2-m levels. Although a correlation with disease stage likely exists, there was a substantial proportion of patients with high β2-m levels already at Binet A stage (low tumor burden). Possibly, CLL cells from these patients are more activated in vivo and shed more abundant β2-m. Taken all the above into consideration, the data indicate that the role of β2-m as a prognostic tool should be re-evaluated possibly in prospective studies involving large patient cohorts.

Report on an anti‐doping operation in Guadeloupe: High number of positive cases and inferences about doping habits

“…Previous studies have shown that it is easily detected in serum and that it is also detectable in urine after strenuous physical exercise or sometimes even without exercise. [2,13] Here, we demonstrate that CERA continues to be attractive for some athletes. The very strong CERA signal identified in serum was probably due to the short time between injection and control.…”

A fast preparative method for detection of recombinant erythropoietin in blood samples

“…Antidoping control of CERA currently relies on screening by enzymelinked immunosorbent assay (ELISA) test [7] and confirmation analysis by immunoaffinity and IEF. [3] During our experience with this process, we became aware of false negative results with ELISA that were clearly positive with IEF. We thus advise IEF for both screening and confirmation, as is the case for detecting other EPOs in urine.…”

“…For this, 1 volume of serum (or plasma) is diluted with 9 volumes of 0.9% NaCl and 5 volumes of 1.8 M perchloric acid are then added. Most of the proteins are immediately precipitated and the supernatant is collected after 10 min of centrifugation at 2700 g. Perchloric acid is then neutralized by slowly adding 5 volumes of 1.8 M KHCO 3 . A second precipitation occurs due to formation of insoluble KClO 4 according to the reaction: HClO 4 + KHCO 3 → H 2 O + KClO 4 + CO 2 .…”

“…While first-and second-generation recombinant EPOs are detectable in urine, we showed that analysis in blood is more appropriate for the thirdgeneration, continuous erythropoietin receptor activator (CERA), due to its poor excretion in urine. [3] However, isoelectric focusing (IEF) of EPO in serum or plasma is not as simple as in urine since it requires an initial preparative step by immunoaffinity chromatography. [4] We thus developed a convenient method (more than 20 samples can be treated in parallel) for preparing plasma or serum samples, making IEF as easy to use in blood as in urine.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…However, its significantly increased plasma half-life has allowed the expansion and transfer of existing analytical approaches (namely isoelectric focusing) from urine to blood, which yielded various adverse analytical findings in 2008. [43] A more rapid complementary immunological assay was developed in 2009, enabling the sensitive analysis of up to 70 samples per day to provide fast indication of whether a specimen is suspicious for CERA and requires further tests. [44] Based on a two-sided enzyme-linked immunosorbent assay (ELISA) directed against EPO as well as polyethylene glycol (PEG), a lower limit of quantification of 20 pg/mL was accomplished, allowing the detection of CERA in serum for more than four weeks after application of a single dose of 200 µg in selected individuals.…”