Recombinant human erythropoietin (rhEPO) remains a persistent threat to racing integrity. As a rapidly eliminated out-of-competition drug, rhEPO use can only be controlled by screening large numbers of samples at high-levels of sensitivity. The sensitivity of a commercial rhEPO ELISA screen was increased four-fold through the use of high-sensitivity streptavidin-Horse Radish Peroxidase (HRP) and the assay specificity improved by competing samples with a monoclonal anti-rhEPO antibody that targets the same epitope as the capture antibody. These two innovations resulted in an ELISA screen for rhEPO with a LOD of 1 mIU/ml. Finally, the ELISA was miniaturised in a 384-well based assay for increased sample throughput with an approximately four-fold reduction in assay costs.
Improved screening sensitivity requires matching increases in sensitivity for mass spectrometry confirmation. rhEPO confirmation was trialled on a range of low and high resolution Thermo mass spectrometers, taking advantage of advances in nano-chromatography to improve analytical performance.