Tohoku J. Exp. Med., 2004, 203 (4)

Characterization of a Shiga Toxin 1-Neutralizing Recombinant Fab Fragment Isolated by Phage Display System

KAZUYUKI INOUE, KUNIHIKO ITOH, HIROSHI NAKAO,¹ TAE TAKEDA^1,* and TOSHIO SUZUKI

Department of Pharmaceutical Science, Akita University Hospital, Akita 010-8543, and ¹Department of Infectious Diseases, National Research Institute for Child Health and Development, Tokyo 154-8567

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Molecular structural analysis of Stx-neutralizing monoclonal antibody (mAb) will be helpful for development of therapeutics and prophylactics for STEC infection. In this study, we cloned the genes of Stx 1-neutralizing mAb, termed 5-5B from hybridoma cells by phage display system and characterized its recombinant Fab (rFab) fragment. 5-5B rFab fragment reacted with Stx1, but not with Stx2 and bovine serum albumin (BSA). It also showed the neutralizing activity against the cytotoxicity of Stx1. These results imply that 5-5B rFab fragment is functionally identical to parent mAb. The variable heavy (V_H) and light domains were found to be highly homologous with the derived germ line sequences. As for V_H domain, the complementarity determining regions (CDRs) showed higher replacement/substitution mutation ratio than that in the frame work regions. Among the regions, CDR2 showed the most frequent nucleotide and amino acid substitutions. These results suggest that heavy chain CDR2 may mainly be associated with the 5-5B function, that is neutralizing cytotoxicity of Stx1.

keywords —— phage display; recombinant fab; shiga toxin 1; monoclonal antibody

© 2004 Tohoku University Medical Press

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Address for reprints: Prof. T. Suzuki, Department of Pharmaceutical Science, Akita University Hospital, 1-1-1 Hondo, Akita 010-8543, Japan.

e-mail: suzuki@hos.akita-u.ac.jp

Footnotes: The nucleotide sequence data reported in this paper are available from DDBJ/EMBL/GenBank nucleotide sequence databases under accession numbers, AB116255-6.
^*Tae Takeda died on May 15, 2001.