Glycoprotein microarray for the fluorescence detection of antibodies produced as a result of erythropoietin (EPO) abuse

Abstract

With the commercial availability of recombinant human erythropoietin (rHuEPO), there is significant scope for athletes, especially those competing in endurance sports, to illicitly enhance their performance by increasing their aerobic capacity through enhanced erythrocyte production and hence oxygen transport. While such abuse has been confirmed in a number of human sports, there is also the possibility that rHuEPO can be abused in animal based sports such as thoroughbred horseracing. The direct detection of rHuEPO abuse, using either urine or blood samples, is challenging as the recombinant glycoprotein is similar to that produced endogenously and typically can only be measured above background levels within 4 days of administration. However, it is known that an immune response occurs when horses are doped with rHuEPO. The production of a specific antibody in response to doping with rHuEPO provides a target analyte that is not only different to endogenous species but one which resides in the body for considerably longer than the glycoprotein itself, significantly extending the measurement window. Here we have developed a glycoprotein microarray which exploits the antibody–antigen interaction to provide a means of detecting rHuEPO abuse in animals through the measurement of erythropoietin (EPO) antibodies (anti-HuEPO antibodies). Three commercially available isoforms of rHuEPO (Eprex^®, Aranesp^® and NeoRecormon^®) were arrayed onto the planar surface of a nitrocellulose-coated microarray slide to act as the capture molecule in the assay. The assay was achieved by incubation of the microarray with solutions containing the anti-HuEPO antibody, followed by incubation with a fluorescently tagged secondary antibody. This ‘sandwich’ based assay enabled the fluorescent based detection of anti-HuEPO antibodies using an array-scanner. The EPO glycoprotein microarray was shown to be specific for anti-HuEPO antibodies. To detect anti-HuEPO antibodies in spiked serum samples an optimal dilution of the serum with buffer of 1 : 4 was established. Using Eprex^®-10,000 IU as the capture molecule, the lowest concentration of anti-HuEPO antibody which was detected using the microarray was 148 pM, suggesting that the developed microarray platform could be used as a screen of EPO abuse.