Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC‐MS/MS and western blotting: a comparative study

Abstract

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening.

A method for confirming CJC‐1295 abuse in equine plasma samples by LC–MS/MS

“…The method detailed here is derived from the rhEPO confirmation method developed by Timms et al It uses an efficient single‐step immunoaffinity purification to separate the CJC‐1295‐protein conjugates from abundant plasma proteins that would otherwise interfere with analysis, followed by tryptic digestion and targeted MS/MS. It should be noted that previous methods that have claimed to detect CJC‐1295 have targeted peptides without the DAC that defines CJC‐1295 .…”

“…1413071.1). 6 2.4 | Preparation of RD4-labeled paramagnetic beads RD4, the monoclonal antibody against CJC-1295, was covalently attached to M-280 Tosyl Activated Dynabeads as described in Timms et al 9 M-280 Tosyl Activated Dynabeads (30 mg/mL) were resuspended by vortexing and 5 mg (165 μL) removed for transfer to a low bind 1.5 mL Eppendorf microfuge tube. Dynabeads were pelleted using a rare-earth magnet for all washing and recovery steps.…”

“…To provide absolute certainty in the identification of CJC‐1295, we have developed a method for confirming the presence of CJC‐1295 in equine plasma by liquid chromatography−tandem mass spectrometry (LC−MS/MS). This method operates in a manner equivalent to other MS‐based confirmation methods, using immunoaffinity capture followed by trypsin digestion and targeted MS/MS to provide confirmation of CJC‐1295 in equine blood at high sensitivity …”

An immuno polymerase chain reaction screen for the detection of CJC‐1295 and other growth‐hormone‐releasing hormone analogs in equine plasma

“…SDS polyacrylamide gel electrophoresis (SDS‐PAGE) and western blotting were performed as described in Timms et al RD4 and RD6 monoclonal antibodies were diluted 1 in 1000 for use. Donkey F (ab’) polyclonal secondary antibody to mouse IgG (HRP conjugated) was used at 1 in 20 000.…”

Screening and confirmatory analysis of recombinant human erythropoietin for racing camels' doping control

“…The detection of rHuEPO usually involves immunoaffinity capture and electrophoretic‐based detection in human and animal subjects or immunoaffinity capture and mass spectrometry (MS) in nonhuman species through the detection of rHuEPO‐specific peptides . Because these peptides are used to discriminate any suspicious sample from real administration cases, analytical methods crucially need both high sensitivity and extensive specificity.…”