Comprehensive evaluation of commercial anti-human EPO antibodies for TD2021EPO

Code: 21B07CR

All detection methods for ERA-doping are currently based on electrophoretic separation and Western blotting and due to its high sensitivity apply the same primary antibody (clone AE7A5 anti-EPO

antibody). Upon initiative of the grant applicant, a biotinylated version of the antibody was generated in 2018, which led to further increase in sensitivity and simplification of the method. The antibody

also became part of WADA TD2021EPO. One drawback of clone AE7A5 antibody is that it non-specifically binds to some proteins, which are present in human urine and blood. The non-specific

binding is caused by the fact, that the peptide used for generating the antibody contained two errors in the amino acid sequence. Hence, all doping control samples have to be first immunoaffinity-purified before electrophoresis. For this purpose, a different EPO antibody has to be used. To ensure better selectivity both the ISL [11] and the TD2021EPO, even if not totally mandatory, recommend that the CP differs from the ITP. Since no alternative to clone AE7A5 antibody is known so far, laboratories have to establish two different immunoaffinity purification procedures for ITP and CP. On the other hand, there are ca. 150 anti-human EPO antibodies commercially available, which have largely not been compared with clone AE7A5 antibody regarding sensitivity and selectivity. If an alternative antibody could be found, this would not only simplify EPO testing but would also generate more flexibility for the ITP and CP. Aim of the proposed project is a large scale evaluation of commercial anti-human EPO antibodies. About 100 antibodies will be tested regarding their sensitivity and selectivity in comparison with clone AE7A5 EPO antibody.

Main findings

164 antibodies were tested regarding their sensitivity in comparison with clone AE7A5 EPO antibody. Out of 70 polyclonal and 94 monoclonal antibodies a clear favourite was identified. It surpassed clone AE7A5 not only by sensitivity but also by specificity. The antibody is a recombinant rabbit monoclonal antibody (clone HL1794) and is worldwide available from the manufacturer (Genetex) as well as large international distributors (e.g. Abcam, ThermoFisher Scientific, Fisher Scientific). The antibody was fully validated for urine using an HRP-labelled and cross-reactivity minimized secondary antibody. It allowed the detection of all ERAs (rEPO, NESP, EPO-Fc, CERA) down to at least 0.1 MRPL of TD2024EPO. Similar results were obtained serum samples. The antibody did also not bind to proteins known to be nonspecifically bound by clone AE7A5 (ZAG, TrxR, NSE, NNE, enolase). Conclusions: Anti-EPO antibody clone HL1794 outperformed the current detection antibody clone AE7A5 regarding sensitivity and specificity. Thus, it is a very promising antibody, which will further improve detection of ERA-doping.