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

Lasne, Françoise; Martin, Laurent; Martin, Jean-Antoine

doi:10.1002/dta.171

Cited by 9 publications

(6 citation statements)

References 7 publications

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“…ethanol, acetonitrile), [24,44,45] polyethylene glycol-containing solutions (e.g. perchloric acid) [47] can selectively enrich EPO and MIRCERA in the supernatant. perchloric acid) [47] can selectively enrich EPO and MIRCERA in the supernatant.…”

Section: Selective Enrichment Of Epo-fc Using Acn Precipitationmentioning

confidence: 99%

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Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing

Reichel

Thevis

2012

Drug Testing and Analysis

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The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc-part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO-Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance-enhancing agents, EPO-Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO-Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO-Fc from human serum via high capacity protein A beads and subsequently detects EPO-Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO-Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS-PAGE, SAR-PAGE) and Western double-blotting with chemiluminescence detection - a method already established in routine EPO anti-doping control. The latter strategy allows the detection of EPO-Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol.

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Section: Selective Enrichment Of Epo-fc Using Acn Precipitationmentioning

confidence: 99%

“…PEG-6000 in saline), [46] and acidification (e.g. perchloric acid) [47] can selectively enrich EPO and MIRCERA in the supernatant. Hence, it was tested whether a precipitation strategy (acetonitrile) might be also useful for enrichment of EPO-Fc in serum.…”

Section: Selective Enrichment Of Epo-fc Using Acn Precipitationmentioning

confidence: 99%

Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing

Reichel

Thevis

2012

Drug Testing and Analysis

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“…Recently, several methods were developed and published for EPO‐specific enrichment using either ELISA‐plates or columns . Another option is EPO‐selective protein depletion by, for example, protein precipitation with perchloric acid, which enriches EPO in the supernatant . Currently, several anti‐doping control labs are using immunoaffinity purification already for EPO‐testing with the regular lower‐throughput standard IEF‐PAGE method (e.g.…”

Section: Resultsmentioning

confidence: 99%

Application of high‐throughput IEF‐PAGE for EPO‐doping testing

Reichel

2012

Drug Testing and Analysis

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Since its first publication in 2000, the isoelectric focusing (IEF) method of erythropoietin (EPO) used in doping control has been considered a procedure with relatively small sample number capacity. To overcome this limitation, a variation of the current protocol was evaluated, which uses double-sized gels with 48-120 wells plus three electrodes and hence multiplies the capacity of the electrophoretic chamber. With this modification up to 120 samples and standards can be run on a single gel - thus, making IEF-PAGE of EPO a high-throughput method. The protocol is ideally suited for large-scale screening purposes.

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“…This is the underlying principle of the only direct method of rhEPO detection that has been approved by the court of arbitration for sport. [6364] It uses electrophoretic techniques to separate the isoform profiles of recombinant and endogenous EPO in the urine according to their isoelectric points. [65] Darbepoetin alpha needs to be administered only once a week and as it has five glycosylation sites.…”

Section: Abuse Of Erythropoietin In Competitive Sportsmentioning

confidence: 99%

Erythropoietin use and abuse

John

Jaison

Jain

et al. 2012

Indian J Endocr Metab

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Recombinant human erythropoietin (rhEPO) is arguably the most successful therapeutic application of recombinant DNA technology till date. It was isolated in 1977 and the gene decoded in 1985. Since then, it has found varied applications, especially in stimulating erythropoiesis in anemia due to chronic conditions like renal failure, myelodysplasia, infections like HIV, in prematurity, and in reducing peri-operative blood transfusions. The discovery of erythropoietin receptor (EPO-R) and its presence in non-erythroid cells has led to several areas of research. Various types of rhEPO are commercially available today with different dosage schedules and modes of delivery. Their efficacy in stimulating erythropoiesis is dose dependent and differs according to the patient's disease and nutritional status. EPO should be used carefully according to guidelines as unsolicited use can result in serious adverse effects. Because of its capacity to improve oxygenation, it has been abused by athletes participating in endurance sports and detecting this has proved to be a challenge.

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

Cited by 9 publications

References 7 publications

Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing

Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing

Application of high‐throughput IEF‐PAGE for EPO‐doping testing

Erythropoietin use and abuse

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