Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library

Jiebo Mi; Jin Yan; Zhenquan Guo; Meiping Zhao; Wenbao Chang
September 2005
Analytical & Bioanalytical Chemistry;Sep2005, Vol. 383 Issue 2, p218
Academic Journal
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 DH5α 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 1.0×108 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.


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