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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, 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.

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

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