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Title: SINGLE CHAIN ANTIBODY WITH SPECIFICITY FOR LISTERIA MONOCYTOGENES

Author
item Paoli, George
item Chen, Chinyi
item Brewster, Jeffrey

Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2004
Publication Date: 6/2/2004
Citation: Paoli, G., Chen, C., Brewster, J.D. 2004. Single chain antibody with specificity for listeria monocytogenes. Journal of Immunological Methods.Vol 289. page 147-155.

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. In this research, antibody phage display was used to produce an antibody fragment 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. Future work will produce specific antibodies for all strains of L. monocytogenes, as well as other food-borne pathogens. This work will allow our laboratory and other researchers to develop much-needed rapid tests for L.monocytogenes, giving producers and regulators a vital tool for controlling food-borne disease.

Technical Abstract: Single chain antibody (scFv) fragments exhibiting specific binding to Listeria monocytogenes strains were isolated from a pool of random scFv fragments expressed on the surface of filamentous bacteriophage. Positive selection (panning) using L. monocytogenes was used to enrich for phage clones with the desired binding affinity, and negative selection using L. innocua and L.ivanovii was used to remove phage expressing cross-reactive antibody fragments. A single phage clone, Lm:A8, was selected using two independent phage panning schemes. A rapid assay was devised to determine phage antibody binding specificity and was used to develop a selectivity profile for individual phage clones. The Lm:A8 clone was screened against a panel bacteria consisting of 8 strains of L. monocytogenes, one each of the other 6 species of Listeria and 9 other relevant species of bacteria. A collection of individual clones from the penultimate phage panning was also screened against a subset of the panel of bacteria. The selectivity profiles indicate that multiple clones, including Lm:A8, exhibit binding to one or more strain of L. monocytogenes without cross-reactivity toward any other species in the panel. This is the first report of a species-specific antibody for viable cells of L. monocytogenes (i.e., the ability to bind to L. monocytogenes without cross-reactivity toward any other species of Listeria).