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United States Department of Agriculture

Agricultural Research Service

Title: Development of Biosensors for Detection of Food-Borne Pathogens by Antibody Phage

Authors
item Paoli, George
item Brewster, Jeffrey

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 25, 2004
Publication Date: October 11, 2004
Citation: Paoli, G., Brewster, J.D. 2004. Development of biosensors for detection of food-borne pathogens by antibody phage. (Meeting Abstract). 68(1):146-149.

Technical Abstract: The development of these immunosensor methods for the detection of foodborne pathogens depends on the availability of antibodies with sufficient specificity. Antibodies have been produced for several food pathogens, and researchers have made good progress in developing rapid tests for those bacteria. However, conventional antibody production methods have failed to produce effective antibodies to several important pathogens, such as Listeria monocytogenes, preventing development of rapid tests There are seven species of Listeria, of which only L. monocytogenes is a human pathogen. Thus, tests for L. monocytogenes must be very specific, since other non-pathogenic species of Listeria are widely distributed in the environment. Phage display has proven a useful tool for the isolation of antibody fragments with desired specificities. The technique involves the display of a library of single-chain antibody (scFv) fragments on the surface of filamentous phage followed by selection of the desired recombinant phage by means of specific binding to an antigen of interest. Although phage display has advantages over conventional polyclonal and monoclonal antibody production, it has not been widely used for the selection of immunoreagents for the detection of food-borne pathogens. We have selected and screened phage displayed scFv fragments to isolate a phage displayed antibody that detects several strains of L. monocytogenes, and does not cross-react with any of the other five species of Listeria. Thus, this antibody fragment shows the high degree of specificity required for accurate detection of L. monocytogenes. As determined by western blot analysis, the antibody binds to polypeptides with apparent molecular masses of 65 kD and 100 kD that are both on the surface of L. monocytogenes cells and exported out of the cells. The efficacy of this antibody for the detection of L. monocytogenes was examined by ELISA using L. monocytogenes grown under a variety of conditions, including growth media commonly used for the isolation of L. monocytogenes from food. The antigen was present on the surface of the cells grown between 20oC and 42oC but was not present on the cell surface when cells were grown at or below15oC. The identity of the polypeptide antigen is being determined in order to take a more rational approach to the development of an antibody-based method for the specific detection of L. monocytogenes.

Last Modified: 7/22/2014