Mi Jiebo, Yan Jin, Guo Zhenquan, Zhao Meiping, Chang Wenbao
Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P R China.
Anal Bioanal Chem. 2005 Sep;383(2):218-23. doi: 10.1007/s00216-005-3401-3. Epub 2005 Sep 13.
The production of a large amount of specific antibodies against erythropoietin (EPO) is necessary for both clinical treatment and doping control. However, the weak immunogenicity of EPO and the side effects of excessive injection make the conventional immunological protocol rather inefficient and time-consuming. In this study, a single-chain antibody fragment of variable region (scFv) against recombinant human erythropoietin (rHuEPO) was produced after three rounds of panning a phage display antibody library. The selected scFv-B2 was expressed in soluble form in Escherichia coli DH5alpha F' and purified by His-bond nickel affinity chromatography with a yield of about 1-2 mg of antibody in 1 L of the culture supernatant. The molecular weight of the scFv was estimated to be 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the affinity constant was found to be 1.0 x 10(8) L mol(-1) based on a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). The potential ability of the scFvs for immunopurification of rHuEPO from related sample was demonstrated by using a double-antibody sandwich ELISA. The reported method is a very powerful tool to produce specific antibodies for rHuEPO detection demands.
生产大量针对促红细胞生成素（EPO）的特异性抗体对于临床治疗和兴奋剂检测都至关重要。然而，EPO的弱免疫原性以及过量注射的副作用使得传统免疫方案效率低下且耗时。在本研究中，经过三轮筛选噬菌体展示抗体库后，产生了针对重组人促红细胞生成素（rHuEPO）的可变区单链抗体片段（scFv）。所选的scFv-B2在大肠杆菌DH5alpha F'中以可溶形式表达，并通过His键镍亲和层析纯化，在1升培养上清液中抗体产量约为1-2毫克。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计scFv的分子量为29 kDa，基于竞争性间接酶联免疫吸附测定（CI-ELISA）发现亲和常数为1.0×10⁸ L mol⁻¹。通过使用双抗体夹心ELISA证明了scFv从相关样品中免疫纯化rHuEPO的潜在能力。所报道的方法是满足rHuEPO检测需求生产特异性抗体的非常强大的工具。
