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Journal of Clinical Microbiology, July 2004, p. 2966-2976, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.2966-2976.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Isolation of Recombinant Antibodies against EspA and Intimin of Escherichia coli O157:H7

Sarah A. Kühne,1,2 William S. Hawes,1 Roberto M. La Ragione,3 Martin J. Woodward,3 Garry C. Whitelam,1 and Kevin C. Gough1,4*

Department of Biology, University of Leicester, Leicester LE1 7RH,1 Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB,3 ADAS Rosemaund, Preston Wynne, Hereford HR1 3PG, United Kingdom,4 Ecole Supérieure de Biotechnologie de Strasbourg, Pôole API, F-67400 Strasbourg, Illkirch, France2

Received 23 January 2004/ Returned for modification 20 March 2004/ Accepted 10 April 2004

Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragment containing amino acid residues 258 to 361. Recombinant intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or intimin. Antibodies recognizing different C-terminal epitopes on intimin bound specifically to the gamma intimin of O157:H7 and not to other classes of intimin. Antibodies recognizing EspA from E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of {gamma} intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.

* Corresponding author. Mailing address: Department of Biology, Adrian Building, University of Leicester, University Rd., Leicester LE1 7RH, Leicestershire, United Kingdom. Phone: 44-116-2231782. Fax: 44-116-2523330. E-mail: kcg3{at}le.ac.uk.

Journal of Clinical Microbiology, July 2004, p. 2966-2976, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.2966-2976.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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