This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deng, X. K.
Right arrow Articles by Morrow, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deng, X. K.
Right arrow Articles by Morrow, K. J., Jr.

 Previous Article  |  Next Article 

Clinical and Diagnostic Laboratory Immunology, July 2003, p. 587-595, Vol. 10, No. 4
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.4.587-595.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Recombinant Single-Chain Variable Fragment Antibodies Directed against Clostridium difficile Toxin B Produced by Use of an Optimized Phage Display System

Xiao K. Deng,1,{dagger} Lance A. Nesbit,2 and K. John Morrow, Jr.1*

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430,1 Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona 857242

Received 21 October 2002/ Returned for modification 25 January 2003/ Accepted 20 March 2003

Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10-8 M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.

* Corresponding author. Present address: Department of Research and Development, Meridian Bioscience, 4571 River Hills Dr., Cincinnati, OH 45244. Phone: (513) 271-3700. Fax: (513) 271-0744. E-mail: JMorrow{at}meridianbioscience.com.

{dagger} Present address: Department of Research and Development, Meridian Bioscience, Cincinnati, Ohio.

Clinical and Diagnostic Laboratory Immunology, July 2003, p. 587-595, Vol. 10, No. 4
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.4.587-595.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lim, D. V., Simpson, J. M., Kearns, E. A., Kramer, M. F. (2005). Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare. Clin. Microbiol. Rev. 18: 583-607 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»