Analytical and Bioanalytical Chemistry

, Volume 383, Issue 2, pp 218–223

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
Original Paper

DOI: 10.1007/s00216-005-3401-3

Cite this article as:
Mi, J., Yan, J., Guo, Z. et al. Anal Bioanal Chem (2005) 383: 218. doi:10.1007/s00216-005-3401-3

Abstract

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.

Keywords

scFv Erythropoietin Phage display antibody library 

Abbreviations

CFU

colony forming units

ELISA

enzyme-linked immunosorbent assay

HRP

horseradish peroxidase

IMAC

immobilized metal affinity chromatography

IPTG

isopropyl-β-d-thiogalactopyranoside

NTA

nitrilotriacetic acid

OVA

ovalbumin

PEG

poly(ethylene glycol)

PBS

0.01 mol L−1 phosphate buffered saline

PBST

0.01 mol L−1 PBS containing 0.05% Tween 20

rHuEPO

recombinant human erythropoietin

scFv

single-chain antibody fragment of variable region

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

TMB

3,3′,5,5′-tetramethylbenzidine

2×TYAG

2×TY medium containing 100 μg mL−1 Ampicillin and 2% glucose

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Jiebo Mi
    • 1
  • Jin Yan
    • 1
  • Zhenquan Guo
    • 2
  • Meiping Zhao
    • 1
  • Wenbao Chang
    • 1
  1. 1.Institute of Analytical Chemistry, College of Chemistry and Molecular EngineeringPeking UniversityBeijing 100871P. R. China
  2. 2.College of Life SciencesPeking UniversityBeijing 100871P. R. China