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The development of anti-DR4 single-chain Fv (ScFv) antibody fused to Escherichia coli alkaline phosphatase

대장균의 alkaline phosphatase가 융합된 anti-DR4 single-chain Fv (ScFv) 항체의 개발

  • Han, Seung Hee (Division of BioHealth Science, College of Natural Sciences, Changwon National University) ;
  • Kim, Jin-Kyoo (Division of BioHealth Science, College of Natural Sciences, Changwon National University)
  • 한승희 (창원대학교 자연과학대학 생명보건학부) ;
  • 김진규 (창원대학교 자연과학대학 생명보건학부)
  • Received : 2016.02.23
  • Accepted : 2016.03.03
  • Published : 2016.03.31

Abstract

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

Keywords

alkaline phophatase;ELISA;immunocytochemistry;single-chain variable domain fragment (ScFv);Western blot

Acknowledgement

Supported by : 창원대학교

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