Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Giménez, Estela; Bolós, Carme de; Belalcazar, Viviana; Andreu, David; Borràs, Eva; Torre, Beatriz G. de la; Barbosa, J.; Segura, Jordi; Pascual, José A.

doi:10.1007/s00216-007-1334-8

Cited by 8 publications

(3 citation statements)

References 24 publications

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“…Three types of analytical techniques are of practical use in doping analysis of EPO drugs. Enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) has been reported for screening analysis of human EPO, − rhEPO, , and CERA. , In addition, a sensitive immunochromatographic assay was proposed for detection of human EPO and its analogues …”

mentioning

confidence: 99%

Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control

et al. 2010

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Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic peptide, T8 ((54)MEVGQQAVEVWQGLALLSEAVLR(76), common to the three proteins), and improved immunoaffinity extraction. The analytes were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000. The extracted analytes were digested by trypsin and analyzed by LC-MS/MS. The limit of identification was 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma; the limit of detection was 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO. Specificity of the method was assessed via BLAST and SEQUEST protein database searches, and the T8 is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO. This method was successful in identifying CERA and DPO in plasma samples collected from research horses post the drug administrations. It provides a useful tool in the fight against blood doping with CERA, rhEPO, and DPO in racehorses. Additionally, the following two technical approaches adopted in this study may also be helpful in protein identifications and biomarker discoveries in a broad scope: precipitating plasma proteins with PEG 6000 to improve immunoaffinity extraction efficiency of the target proteins and making a large and more lipophilic peptide detectable at low concentrations by increasing its solubility in the sample solvent.

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confidence: 99%

Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control

et al. 2010

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“…To estimate the amount of xl EPO muteins in the COS-1 supernatant, we performed a densitometry analysis of Western blots. In Western blot analysis, the recognition of hyperglycosylated huEPO analogs by an anti-huEPO sequence antibody was reported to be weak compared with huEPO recognition, and was improved by the partial elimination of carbohydrate chains [ 41 ]. Therefore, we used Western blots of deglycosylated samples ( Fig 2A , bottom panel ) for densitometry analysis, because the added N -linked carbohydrates may prevent the anti- xl EPO sequence antibody from binding to the xl EPO muteins.…”

Section: Resultsmentioning

confidence: 99%

The Influence of Artificially Introduced N-Glycosylation Sites on the In Vitro Activity of Xenopus laevis Erythropoietin

et al. 2015

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Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1–3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR.

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“…The monoclonal anti-hEPO antibody clone 9C21D11 and overnight incubations were used throughout the study in order to maximize the capacity of the method. Attempts to use the monoclonal antihEPO antibody clone AE7A5 (used in the current IEF method for hEPO as it has shown to have the best sensitivity for blotting purposes [16,17]) or the polyclonal anti-hEPO antibody (AB-286-NA), in the selected conditions, resulted in a much lesser sensitivity (data not shown). This is not surprising since, clone AE7A5 had already shown to perform very badly when immobilised on a surface [18].…”

Section: Iap Purification Methods Developmentmentioning

confidence: 97%

Purification of erythropoietin from human plasma samples using an immunoaffinity well plate

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Llop

Bolós

et al. 2010

Journal of Chromatography B

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Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Cited by 8 publications

References 24 publications

Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control

Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control

The Influence of Artificially Introduced N-Glycosylation Sites on the In Vitro Activity of Xenopus laevis Erythropoietin

Purification of erythropoietin from human plasma samples using an immunoaffinity well plate

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