Detection of isoforms of recombinant human erythropoietin by various plant lectins after isoelectric focusing.
A screening method to determine the binding behavior of lectins toward recombinant human erythropoietin (rHuEPO) was developed. Twenty-three different lectins were tested for this purpose. rHuEPO isoforms were separated by isoelectric focusing using the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) accredited method for the direct detection of the prohibited doping substance erythropoietin (EPO). For the visualization of the rHuEPO isoforms lectins were used instead of antibodies. Optimization of the screening protocol enabled the detection of a maximum number of rHuEPO isoforms. By means of this protocol information about the binding properties of a lectin toward each individual rHuEPO isoform was accessible. All evaluated lectins showed significant differences in their binding behavior. The most intense response was obtained with WGA, DSL, PHA-E, LEL, PSA, and LCA. While WGA, DSL, PHA-E, and LEL were able to bind all isoforms detected by the standard antibody, LCA and PSA demonstrated a clear preference for rHuEPO isoforms located in the more basic region of the electropherogram. Further lectins tested were ConA, succWGA, PHA-L, RCA, SNA, MAA, STL, ECL, GSL-II, SJA, SBA, UEA-I, Jacalin, PNA, DBA, GSL-I, and VVA. Compared to the lectins mentioned above, they showed reduced sensitivity. Endogenous and recombinant EPO only differ in the composition of their N- and O-glycan moieties. As lectins possess the unique ability to recognize subtle differences in glycan substructures, they represent an interesting approach for their structural characterization. Furthermore, they might be useful for affinity enrichment/purification of rHuEPO in doping control.
Nagano
Marietta
M
Doping Control Laboratory, ARC Seibersdorf Research GmbH, A-244 Seibersdorf, Austria.
Stübiger
Gerald
G
Marchetti
Martina
M
Gmeiner
Günter
G
Allmaier
Günter
G
Reichel
Christian
C
D004300:Doping in Sports; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007525:Isoelectric Focusing; D037121:Plant Lectins / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins
D037121:Plant Lectins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004921:Erythropoietin