Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Abstract

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87-90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81-95), NESP (81-95), and NESP (86-104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide-OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81-95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84-106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86-91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.