Giménez E, de Bolós C, Belalcazar V, Andreu D, Borrás E, De la Torre B G, Barbosa J, Segura J, Pascual J A
Departament de Química Analítica, Universitat de Barcelona, Diagonal 647, 08028 Barcelona, Spain.
Anal Bioanal Chem. 2007 Aug;388(7):1531-8. doi: 10.1007/s00216-007-1334-8. Epub 2007 May 30.
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.
促红细胞生成素（EPO）是一种调节红细胞生成的激素。重组人促红细胞生成素（rHuEPO）和新型促红细胞生成刺激蛋白（NESP）已被生产用于治疗目的，也用于提高运动成绩。rHuEPO和NESP的一级序列仅相差五个氨基酸。由于高度同源，到目前为止尚未获得能够区分这两种分子的抗体。本研究的目的是设计与EPO和NESP之间不同的序列（87 - 90aa）相对应的合成肽，然后将其用作免疫原，以开发用于选择性检测EPO和NESP的特异性兔多克隆抗体。合成了三种肽：EPO（81 - 95）、NESP（81 - 95）和NESP（86 - 104），并分别将它们与钥孔血蓝蛋白（KLH）和卵清蛋白（OVA）偶联用于免疫和筛选。通过酶联免疫吸附测定（ELISA）在合成肽 - OVA偶联物上检测所获得的血清，并通过针对相应合成肽的免疫亲和色谱法进行纯化。通过ELISA、十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和等电聚焦对特异性纯化抗体进行表征，随后进行蛋白质印迹分析。针对EPO（81 - 95）产生的抗血清识别rHuEPO但不识别NESP。相反，抗NESP（84 - 106）血清仅在NESP（86 - 91）柱上进行免疫亲和纯化后才产生特异性抗NESP反应。通过选择合适的合成肽可以实现产生针对EPO和NESP的特异性抗体的有效策略。所获得的抗体能够区分rHuEPO和NESP，并且在治疗和反兴奋剂背景下的筛选目的中可能特别有用。
