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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED BIOSENSOR-BASED PROCESSES FOR MULTIPATHOGENIC ANALYTE DETECTION Title: Innovative applications of bacteriophages in rapid detection and identification of foodborne pathogens

Author
item Paoli, George

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 28, 2008
Publication Date: August 6, 2008
Citation: Paoli, G. 2008. Innovative applications of bacteriophages in rapid detection and identification of foodborne pathogens. Meeting Abstract.

Technical Abstract: Relative to traditional microbiological approaches, biosensors are a rapid method for foodborne bacterial pathogen detection. Biosensors function by detecting the interaction of the target pathogen, or pathogen derived molecule, with a biological recognition component which must have sufficient affinity and specificity. In the case of immunosensors, this specificity is imparted by antibodies. Antibody phage display is a molecular method for the selection of antibody fragments of desired specificity and is an alternative to traditional means for the generation of polyclonal or monoclonal antibodies. Antibody phage display involves the cloning of nucleotide sequences encoding the antibody variable regions and display of the antibody fragments as fusion proteins on the surface of bacteriophage. Single chain antibody fragments (scFvs) of desired specificity are selected from a large collection of bacteriophage expressing unique scFvs through the binding of the phage displayed scFv with the target of interest. Immunosensor-based detection methods are available for a number of food-borne pathogens, but, until recently, their application to the detection of L. monocytogenes has been hampered by the lack of species-specific polyclonal serum or monoclonal antibodies. We isolated a L. monocytogenes-specific scFv from a phage display library. A plasmid vector was constructed that allowed the expression of biotinylated scFvs in Escherichia coli. The biotinylated scFvs were purified and coupled to streptavidin-coated magnetic beads. The anti-L. monocytogenes scFv-IMBs exhibited higher efficiencies and improved specificity of capture for L. monocytogenes than were observed for commercially available anti-Listeria IMBs. The anti-L. monocytogenes IMB capture was combined with microbiological and PCR methods for specific detection of L. monocytogenes. In addition, a surface plasmon resonance biosensor was developed using bacteriophage displaying the anti-L. monocytogenes scFv. Specificity of the sensor was demonstrated using common foodborne bacteria and a control phage lacking the scFv. This study demonstrates the applicability of antibody fragments selected via phage display for the development of immunoreagents and the detection of foodborne pathogens.

Last Modified: 10/22/2014
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