Identification of zinc‐alpha‐2‐glycoprotein binding to clone AE7A5 antihuman EPO antibody by means of nano‐HPLC and high‐resolution high‐mass accuracy ESI‐MS/MS

The detection of doping with recombinant erythropoietins (Epo) by isoelectric focusing (IEF) and Western double blotting strongly relies on the specificity of the detection antibody used. Currently a monoclonal mouse antibody (clone AE7A5) is used for that purpose. Despite its excellent sensitivity (amol range) the antibody shows some nonspecific binding behavior. However, the binding occurs outside the currently used pH range for evaluating erythropoietin IEF profiles. A shotgun proteomics approach is described consisting of preparative IEF on large-sized carrier ampholyte gels (pH 3-5), SDS-PAGE, Western single and double blotting, on-membrane elution of intact proteins, on-membrane and in-solution tryptic digestions, as well as nano-HPLC peptide separation and high-resolution high-mass accuracy ESI-MS/MS peptide sequencing. The nonspecifically interacting protein could be identified as zinc-alpha-2-glycoprotein (ZAG). Confirmation analyses were performed using recombinant ZAG (rhZAG) and a monoclonal anti-ZAG antibody. It could be demonstrated that the binding of the monoclonal antihuman EPO antibody (clone AE7A5) to ZAG occurs in a highly concentration-dependant manner and that only samples containing increased amounts of urinary ZAG lead to a detectable interaction of the AE7A5 antibody on Epo-IEF gels.