Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test

Abstract

Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg(-1) day(-1) to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2-5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).

Detection of EPO injections using a rapid lateral flow isoform test

“…Three of the subjects will be positive for doping (10,11,13) being outside the 99.99% CL. Three of the subjects might be suspected for doping (9,12,15) being outside the 99% CL, while only two subjects are negative (25%). The haemoglobin and haematocrit values exceed the limits for competition exclusion only for one subject (13).…”

“…The EPO WGA MAIIA method has previously proven to be successful in the detection of several isoforms of EPO [11] like epoetin alpha, omega, delta, darbepoetin alpha and CERA. The method has also successfully been identifying identified the injection of epoetin-alpha and darbepoetin alpha in horses [15].…”

“…The isoform methods rely on that large numbers of samples from a reference population have been tested to select the most suitable cut-off limits for achieving highest possible specificity and sensitivity for detecting a potential doping misuse with rhEPO. With the One of the recently presented developed EPO WGA MAIIA test kit [11,15,16]EPO isoform methods show quantitative values for EPO isoforms are obtained, the EPO WGA MAIIA test kit [11,15,16], by using percentage of migrated isoforms (PMI) as unit. Quantification allows the use of confidence limits (CL) calculated from the results for the reference population, and makes between method comparison possiblepossible.…”

Patients with anaemia can shift from kidney to liver production of erythropoietin as shown by glycoform analysis

“…A study measuring the interaction between EPO carbohydrate structures and lectins found that the lectin wheat germ agglutinin was especially useful for distinguishing recombinant and endogenous EPO [19]. The interaction between EPO and WGA has been used in a rapid lateral flow test for identification of rhEPO doping in humans and in horses [20,21].…”

Efeito da idade e sexo sobre a concentração sérica de eritropoietina em equinos da raça Árabe

“…A rhEPO também pode ser determinada pela cromatografia liquida acoplada a espectrometria de massa (LC-MS/MS) (Scarth et al 2011). Mais recentemente, o método de micro-cromatografia WGA MAIIA foi desenvolvido para detecção de agentes estimulantes da eritropoiese na urina e plasma de equinos (Lönnberg et al 2012).…”