Screening and confirmation of recombinant human erythropoietin and darbepoietin-α in spiked plasma samples from drug-free horses

Shveta Gupta, Abby Sage, Ashok K Singh

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Methods for screening and confirmation of erythropoiesis stimulating glycopeptides (ESGs) such as endogenous equine erythropoietin (eEPO), recombinant human erythropoietin (rhEPO) and darbepoietin-α (DAR) have been described. Four rhEPO immunoassay kits (R&D ELISA, Diagnostic Systems Limited (DSL) ELISA, chemiluminescent IMMULITE (CHEM), and DiaSorin RIA kits) were evaluated for eEPO, rhEPO and DAR screening in basal and spiked horse samples. The R&D-ELISA detected rhEPO and DAR, but did not detect eEPO. DSL-ELISA, CHEM and DS-RIA kits detected all three ESGs. The immunoassay kits did not differentiate rhEPO and DAR. Deglycosylation of the ESGs increased the detection limits of the immunoassays. DAR, rhEPO and eEPO were extracted from positive or spiked samples by using the Affi-gel and/or immunoaffinity columns. The R&D antibodies did not retain eEPO but retained rhEPO and DAR. The antibodies from DS and DSL effectively retained all three ESGs. The isolated ESGs were confirmed by using either gel electrophoresis or MALDI-TOF-MS methods. DAR and rhEPO extracted from horse plasma were hydrolyzed using trypsin and the fragments were analyzed by the MALDI-TOF-MS for their amino acid sequence. DAR and rhEPO could be differentiated according to their intact mass-ions (m/z 29,000 for rhEPO and m/z 36,000 for DAR) or trypsin-hydrolysis products (m/z 2689 and 2359 for rhEPO and m/z 2696 and 2293 for DAR).

Original languageEnglish (US)
Pages (from-to)96-109
Number of pages14
JournalAnalytica Chimica Acta
Volume552
Issue number1-2
DOIs
StatePublished - Nov 3 2005

Keywords

  • Darbepoietin
  • Erythropoietin
  • MALDI-TOF

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