Iso-electric focusing (IEF) was the first method established to discriminate 
endogenous and recombinant erythropoietins (rEPOs). It is still approved by the 
World Anti-Doping Agency (WADA) as an initial testing procedure to detect 
erythropoiesis stimulating agents (ESAs) in doping control samples. However 
EPO-Fc, one of the prohibited rEPOs designated by WADA, is not detectable with 
the actual IEF conditions. Other newly developed ESAs - luspatercept and 
sotatercept, both activin receptor type II-Fc fusion proteins (ActRII-Fc) - are 
also now prohibited and could be used in combination with rEPOs. Methods of 
identification of ActRII-Fc in blood by SAR/SDS-PAGE have been described, but 
not by IEF. Here we detail improvements in blood sample preparation and IEF 
analysis: A combined immuno-purification of EPOs and ActRII-Fc proteins in a 
single procedure, an appropriate isoforms separation for all proteins using new 
pre-loading and gel conditions, and a single detection of all rEPOs and 
ActRII-Fc proteins after successive incubation with anti-EPO and anti-ActRII 
antibodies. With these changes, distinctive profiles for all the ESAs were 
obtained by IEF. Therefore, IEF could be used as a screening method to detect a 
wide spectrum of prohibited ESAs in blood samples prior to specific confirmation 
for the identified rEPO or ActRII-Fc.