Isolation and characterization of an anti-recombinant erythropoietin single-chain antibody fragment using a phage display antibody library.
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.
Mi
Jiebo
J
Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P R China.
Yan
Jin
J
Guo
Zhenquan
Z
Zhao
Meiping
M
Chang
Wenbao
W
D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001435:Bacteriophages; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005820:Genetic Testing; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008970:Molecular Weight; D019151:Peptide Library; D011994:Recombinant Proteins
D000888:Antibodies, Anti-Idiotypic; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D004921:Erythropoietin