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Journal of Biochemistry Advance Access published online on October 8, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp151
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxcytidine

Motohiro Ohshima, Tomomi Tadakuma, Hideki Hayashi, Kazuyuki Inoue and Kunihiko Itoh*

Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan

*Corresponding Author: Kunihiko Itoh Tel & Fax: +81-54-264-5673 Email: itohk{at}u-shizuoka-ken.ac.jp

Received July 11, 2009; Accepted September 8, 2009


  Abstract

We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly4-Ser)3. The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by ELISA. The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m5dCyd and weakly with 5-methylcytidine (m5Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC50 0.054 mg/ml) was approximately 80 times higher than that of the parent MoAb (IC50 4.27 µ g/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m5dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3, and MDA-MB-453 contained approximately 1% of methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3, and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

Key Words: DNA methylation, monoclonal antibody, scFv, phage display, inhibition ELISA


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