SDS‐PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control

Reichel, Christian; Kulovics, Ronald; Jordan, Veronika; Watzinger, M.; Geisendorfer, Thomas

doi:10.1002/dta.10

Cited by 88 publications

(124 citation statements)

References 22 publications

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“…Three of the subjects will be positive for doping (10,11,13) being outside the 99.99% CL. Three of the subjects might be suspected for doping (9,12,15) being outside the 99% CL, while only two subjects are negative (25%). The haemoglobin and haematocrit values exceed the limits for competition exclusion only for one subject (13).…”

Section: Discussionmentioning

confidence: 99%

“…The basic approach to analytically distinguish between different EPOs is to combine electrophoretic or chromatographic separations of EPO isoforms with sensitive anti-EPO antibody-based detection methods. Endogenous and recombinant EPO glycosylation can be distinguished by charge [5,6], isoelectric point [7], molecular mass [8,9] or by interaction with specific lectins [10,11] and the methods are described in a recent summary [12]. The methods differ in how well they distinguish certain types of glycosylation (the EPO isoform resolution), and by the minimum required amount of EPO for the analysis (the EPO detection sensitivity).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Detection of EPO injections using a rapid lateral flow isoform test

Lönnberg

Lundby

2013

Anal Bioanal Chem

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Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7-21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10-to 20-fold higher analytical rate than what is possible today. days rhEPO was injected every second day during the first 14 days of the study and the method proved to be very effective (xx out of xx) in detecting thisdoping infor the concomitantly obtained samples collected prior to injection. Samples obtained 14 and 21 days after the last injection showed no detectable rhEPO. Seven days after the last injection three positive (>99.99% CL) subjects were found with the test kit. When using 99% CL as cut-off limit, six of the eight subjects (75%) were suspectedfound to be suspicious for doping.Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO.A previous study using indirect methods to determinefor detecting EPO doping on the same samples indicated only that two of the subjects hadwere suspicious valuespositive during 7-21 days after the last injection.We proposesuggest implementing the easy to-use EPO WGA MAIIA rapid lateral flow test as an initial screening procedure in anti-doping work to 1) increase the detection rate of potential rhEPO doping athletes and 2) allow for analysis of a ten to twenty folds higher analytical rate than what is possible todaymore samples than what is possible today.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Detection of EPO injections using a rapid lateral flow isoform test

Lönnberg

Lundby

2013

Anal Bioanal Chem

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“…For EPO anti-doping analysis, the immunoaffinity chromatography needs not only give the highest recovery but also the lowest contamination and the elution buffer has to be suitable for the subsequent SDS-PAGE or IEF analysis. Previous works have asserted that LDS and CHAPS as the most suitable sample buffers for SDS-PAGE and IEF, respectively, thus these buffers were used to elute EPO from the anti-EPO micro well plate (Reihlen et al, 2012;Reichel et al, 2009). However, the reasons and justification for such a claim was unclear.…”

Section: Discussionmentioning

confidence: 99%

“…Reihlen et al (2012) has mentioned that immunoaffinity purification of urine samples is able to improve signal to noise ratio and avoid cross-reactivity. There are quite a number of EPO immunoaffinity purification techniques or kits that have been introduced in the market (Guan et al, 2008;Lonnberg et al, 2010;Reihlen et al, 2012;Lasne et al, 2007;Reichel et al, 2009), which include the anti-EPO monolith column and the customized anti-EPO micro well plate that are widely used in many anti-doping laboratories.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Methods for Purifying Erythropoietin from Urine using Immunoaffinity Applications

Kwan¹,

Baharudin²,

Salim³

et al. 2015

International J. of Biological Chemistry

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Erythropoietin (EPO), a hormone that regulates the synthesis of red blood cells, is frequently abused by athletes. Sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE) is an essential analytical technique in all anti-doping laboratories in order to detect the abuse of EPO. An immunoaffinity purification step is now considered essential for the pre-treatment of urine samples to isolate EPO prior to gel electrophoresis. In this study, we have compared the performance of two immunoaffinity purification techniques in EPO anti-doping analysis, i.e., the anti-EPO micro well plate and anti-EPO monolith column. The anti-EPO monolith column is efficient in removing undesirable proteins except for the Bovine Serum Albumin (BSA) as seen on SDS-PAGE. The BSA was eventually removed from the protocol and the undesirable protein band was eliminated without affecting the performance of the method. Throughout the study, the anti-EPO monolith column emerged as a better option, as it provided a higher sensitivity and higher throughput analysis when compared to the anti-EPO micro well plate. The anti-EPO monolith column has shown consistent results with the EPO recovery rate of 72%, while the limit of detection is as low as 0.5 mIU mLG 1 .

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“…[45] The presented approach highlighted the feasibility of separating endogenous and exogenous EPO by size and band shape as well as the advantage that the SDS-PAGE-based analysis of EPO is not influenced by a so-called 'active' urine or effort-type alteration. The latter phenomenon in particular was the subject of recent studies where supramaximal short-duration exercise was shown to transform typical urinary EPO patterns (as derived from isoelectric focusing) into atypical profiles which, however, did not fulfil the criteria for adverse analytical findings.…”

Section: Erythropoietinmentioning

confidence: 99%