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DOI: 10.1055/s-2005-873188
© Georg Thieme Verlag KG Stuttgart · New York
Construction and Expression of a Single Chain Fv Fragment against Pharmacologically Active Paeoniflorin in Escherichia coli, and its Potential Use in an Enzyme-Linked Immunosorbent Assay
Publication History
Received: March 9, 2005
Accepted: June 22, 2005
Publication Date:
05 January 2006 (online)
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Abstract
A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (VH) and light (VL) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly4Ser)3 linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 μg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.
Key words
Single chain variable fragment (scFv) - paeoniflorin - albiflorin - enzyme-linked immunosorbent assay (ELISA)
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References
- 1 Fitzpatrick J, Fanning L, Hearty S, Leonard P, Manning B M, Quinn J G. et al . Applications and recent developments in the use of antibodies for analysis. Anal Lett. 2000; 33 2563-609
- 2 Laroche Y, Demaeyer M, Stassen J M, Gansemans Y, Demarsin E, Matthyssens G. et al . Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal-antibody specific for human fibrin fragment D-dimer. J Biol Chem. 1991; 266 16 343-9
- 3 Worn A, Pluckthun A. Different equilibrium stability of ScFv fragments: identification, classification, and improvement by protein engineering. Biochemistry. 1999; 38 8739-50
- 4 Coia G, Hudson P J, Irving R A. Protein affinity maturation in vivo using E. coli mutator cells. J Immunol Methods. 2001; 251 187-93
- 5 Winkler K, Kramer A, Kuttner G, Seifert M, Scholz C, Wessner H. et al . Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody. J Immunol. 2000; 165 4505-14
- 6 Robin S, Petrov K, Dintinger T, Kujumdzieva A, Tellier C, Dion M. Comparison of three microbial hosts for the expression of an active catalytic scFv. Mol Immunol. 2003; 39 729-38
- 7 Eto J, Suzuki Y, Ohkawa H, Yamaguchi I. Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants. FEBS Lett. 2003; 550 179-84
- 8 Putalun W, Taura F, Qing W, Matsushita H, Tanaka H, Shoyama Y. Anti-solamargine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants. Plant Cell Rep. 2003; 22 344-9
- 9 Vaughan T J, Williams A J, Pritchard K, Osbourn J K, Pope A R, Earnshaw J C. et al . Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol. 1996; 14 309-14
- 10 Moghaddam A, Borgen T SJ, Kausmally L, Simonsen B, Marvik O J, Brekke O H. et al . Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J Immunol Methods. 2003; 280 139-55
- 11 Lu Z H, Morinaga O, Tanaka H, Shoyama Y. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. Biol Pharm Bull. 2003; 26 862-6
- 12 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 680-5
- 13 Weiler E W, Zenk M H. Radioimmunoassay for determination of digoxin and related compounds in Digitalis lanata . Phytochemistry. 1976; 15 1537-45
- 14 Friguet B, Chaffotte A F, Djavadi-Ohaniance L, Goldberg M E. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 1985; 77 305-19
- 15 Tsumoto K, Shinoki K, Kondo H, Uchikawa M, Juji T, Kumagai I. Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent-application to a human single-chain Fv fragment. J Immunol Methods. 1998; 219 119-29
- 16 Kim S H, Schindler D G, Lindner A B, Tawfik D S, Eshhar Z. Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies. Mol Immunol. 1997; 34 891-906
- 17 Ren X J, Gao S J, You D L, Huang H L, Liu Z, Mu Y. et al . Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency. Biochem J. 2001; 359 369-74
Hiroyuki Tanaka
Department of Medicinal Resources Regulation
Graduate School of Pharmaceutical Sciences
Kyushu University
3-1-1 Maidashi
Higashi-ku
Fukuoka 812-8582
Japan
Phone: +81-92-642-6668
Fax: +81-92-642-6668
Email: htanaka@phar.kyushu-u.ac.jp
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