Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/23/2006
Publication Date: 3/1/2007
Citation: Paoli, G., Brewster, J.D. 2007. Identification of the surface antigen recognized by a listeria monocytogenes-specific phage-displayed antibody fragment and its presence in different physiological conditions. Journal of Rapid Methods and Automation in Microbiology. Vol. 4(1):74-83. Interpretive Summary: Food production facilities and regulatory inspectors need sensitive and accurate tests for the detection of harmful bacteria (pathogens) in food and on food processing surfaces. Rapid tests are essential to allow detection of contaminated foods before they are distributed to consumers. Rapid tests require antibodies (the same molecules produced in the immune system to clear infectious agents from the body) that bind tightly and specifically to the target pathogen. 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. L.monocytogenes is of particular interest, since it is able to grow at refrigerator temperatures and has a high fatality rate in infected individuals. Tests for L. monocytogenes must be very specific, since other, non-harmful species of Listeria are widely distributed in the environment. We are investigating a new approach, antibody phage display, to develop antibodies to food-borne pathogens. We recently selected a phage displayed antibody that could detect several strains of L. monocytogenes, and did not cross-react with any of the other five species of Listeria. This is the first antibody ever isolated to show the high degree of specificity required for accurate detection of L.monocytogenes. In this work the efficacy of this antibody for the detection of L. monocytogenes was examined by growing the bacteria under a variety of conditions, including growth media commonly used for the isolation of L. monocytogenes from food. The target that is detected by the antibody was found to be a protein that is both on the surface of L. monocytogenes cells and exported out of the cells. This information will allow a more rational approach for the production of specific antibodies for the detection of L. monocytogenes, as well as other food-borne pathogens. In turn, the selection of specific antibodies will allow our laboratory and other researchers to develop much-needed rapid tests for L.monocytogenes and other food-borne pathogens, giving producers and regulators a vital tool for controlling food-borne disease.
Technical Abstract: There are six species of Listeria, of which only L.monocytogenes is a human pathogen. Outbreaks of listeriosis due to contaminated foods underscore the need for rapid and specific detection of L.monocytogenes. The development of rapid methods for the detection of L. monocytogenes in food has been hampered by the lack of polyclonal serum or monoclonal antibodies that can specifically detect L. monocytogenes. Phage display has proven a useful tool for the isolation of antibody fragments with desired specificities. We recently selected a phage displayed single-chain antibody that detects several strains of L. monocytogenes, and does not cross-react with any of the other five species of Listeria. The efficacy of this antibody for the detection of L.monocytogenes was examined by ELISA using L. monocytogenes grown at different temperatures and in a variety media commonly used for the isolation of L. monocytogenes from food. The best results were observed when cells were grown in supplemented Fraser enrichment broth. The antigen detected by the phage antibody was present on the surface of the cells grown between 20 degrees C and 42 degrees C but was not present on the cell surface when cells were grown at or below 15 degrees C. As determined by western blot analysis, the antibody bound to polypeptides with apparent molecular masses of 65 kD and 120 kD 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.