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- W1526251012 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." @default.
- W1526251012 created "2016-06-24" @default.
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- W1526251012 date "2007-01-01" @default.
- W1526251012 modified "2023-09-23" @default.
- W1526251012 title "Recombinant antibodies against Clostridium difficile toxin A" @default.
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