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Recombinant antibodies against Clostridium difficile toxin A

Alkhalifah, Mohammed Ali (2007) Recombinant antibodies against Clostridium difficile toxin A. PhD thesis, University of Glasgow.

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Abstract

The carboxy-terminal region of Clostridium difficile Toxin A was expressed as a recombinant protein (ToxinA-CT), purified, and shown to behave as would be expected of the receptor-binding domain. ToxinA-CT was then used to isolate scFv antibodies from a phage display library using conventional bio-panning methods. Progressive enrichment of ToxinA-CT-specific scFvs through 3 rounds of selection was observed. Those scFvs that showed strongest reaction with ToxinA-CT in ELISA were sequenced, expressed as soluble antibodies and purified. A panel of diverse scFvs was established that appeared to bind to non-overlapping epitopes on ToxinA-CT. To assay for protective activity, scFvs were mixed with native Toxin A, added to cells in culture, and the response monitored over a 2 hour period. The scFvs were added to consistently delay the cytopathic activity of Toxin A but were unable to match the neutralizing activity of a polyclonal serum or its Fab derivatives. The epitopes recognised by the scFvs were localized using a sub-cloning strategy. Defined parts of the ToxinA-CT reading frame were recovered by PCR and fusions created with maltose binding protein. scFvs were tested for recognition of the maltose binding fusion proteins. In one instance, a scFv reacted with multiple fusion proteins suggesting recognition of a repeated peptide motif. A maltose binding protein fusion carrying a candidate sequence was successfully recognised by this scFv. Overall, phage display enabled assembly of a panel of scFv antibodies against epitopes in the carboxy-terminal domain of Toxin A. The inability of these antibodies to block the activity of Toxin A may be due to the multivalent interaction between the toxin and its receptor. Alternatively, the ToxinA-CT used in scFv isolated may lack functions that are crucial for receptor interaction and hence potential targets for antibody-mediated inhibition.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Supervisor's Name: Aitken, Dr. Robert
Date of Award: 2007
Depositing User: Elaine Ballantyne
Unique ID: glathesis:2007-2156
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 04 Oct 2010
Last Modified: 10 Dec 2012 13:52
URI: http://theses.gla.ac.uk/id/eprint/2156

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