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J Microbiol Methods. 2011 Jan;84(1):94-100. doi: 10.1016/j.mimet.2010.11.006. Epub 2010 Nov 21.

A human single-chain variable fragment targeting to Vibrio vulnificus RtxA toxin.

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  • 1Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan.

Abstract

Vibrio vulnificus secretes a multifunctional cytotoxin RtxA (VvRtxA), which plays a major role in the bacterial pathogenesis. The lack of an efficient VvRtxA detection tool has hampered the progress of V. vulnificus pathogenesis research. This study aims to isolate VvRtxA specific single-chain variable fragments (scFv) to serve as a detection agent. The VvRtxA C-terminal Gly-Asp (GD) repeat-containing region, which has been implicated for calcium binding and target cell recognition, was chosen as an antigen to screen a scFv phage display library. A scFv clone that reacted positively to VvRtxA was successfully obtained. Using the isolated scFv, a cell-based enzyme-linked immunosorbent assay was established for detecting cell-associated VvRtxA toxin in V. vulnificus infected HEp-2 cells. The result is consistent with previous observations that secretion of VvRtxA toxin is time-dependent on bacteria contacting with host cells. Utilization of scFv for VvRtxA toxin detection provides an applicable strategy devoid of conventional immunization.

Copyright © 2010 Elsevier B.V. All rights reserved.

PMID:
21081141
[PubMed - indexed for MEDLINE]

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