Comprehensive Evaluation of Commercial Antibodies for High Sensitivity Detection of EPO-Doping on Western Blots

Current gel-electrophoretic assays for detecting erythropoietin (EPO) and its analogs depend on the high analytical sensitivity of the anti-EPO antibody clone AE7A5. Consequently, AE7A5 is specified in WADA Technical Document TD2024EPO as the primary antibody recommended to be used on Western blots. However, the TD also permits the usage of "other anti-human EPO antibodies with similar specificity and sensitivity characteristics provided they have been duly validated." So far, a validated alternative to AE7A5 with comparable performance for the Western blot is lacking. To address this gap, we conducted a large-scale evaluation of commercially available anti-human EPO antibodies. A total of 164 antibodies (69 polyclonal, 95 monoclonal) were screened for sensitivity relative to AE7A5. The recombinant rabbit monoclonal clone HL1794 emerged as the preferred candidate, outperforming AE7A5 in both sensitivity and specificity. The antibody was further characterized in urine matrices. All four tested EPO receptor agonists (BRP-EPO, NESP, EPO-Fc, and CERA) were detectable in 10-mL urine samples at concentrations down to at least 0.1× the minimum required performance levels (MRPLs) specified in TD2024EPO. Non-specific binding remained minimal despite the use of a secondary detection antibody. Two commercial conjugation formats of HL1794 were also evaluated: HRP conjugation reduced analytical sensitivity relative to the unconjugated clone, whereas biotinylation produced a substantially elevated background signal. The presented findings are highly promising and represent a valuable advancement for electrophoretic ERA detection methodologies. Additional interlaboratory validation studies may further strengthen the future implementation of HL1794 in routine anti-doping applications.