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Title: DEVELOPMENT OF LISTERIA MONOCYTOGENES-SPECIFIC IMMUNOMAGNETIC BEADS USING A SINGLE-CHAIN ANTIBODY FRAGMENT

Author
item Paoli, George
item Kleina, Lynn
item Brewster, Jeffrey

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2006
Publication Date: 1/10/2007
Citation: Paoli, G., Kleina, L.G., Brewster, J.D. 2007. Development of listeria monocytogenes-specific immunomagnetic beads using a single-chain antibody fragment. Foodborne Pathogens and Disease. 4:74-83

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. Traditional microbiologically based methods for detection of food-borne pathogens often take several days to complete; thus, more 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 so that the pathogen can be isolated or detected from food. 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 the 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 recently used a new approach, antibody phage display, to isolate the first antibodies that react specifically to L. monocytogenes. In this work, we linked these antibodies to microscopic magnetic beads to allow the use of magnets to specifically capture L. monocytogenes from the complex mixture of bacteria found in food. This is the first step in developing a rapid method for the specific detection of L. monocytogenes from food.

Technical Abstract: A method for coupling single-chain antibody fragments (scFvs) to immunomagnetic beads (IMBs) was developed and evaluated using scFvs specific for Listeria monocytogenes. A plasmid vector, pBAD380, was constructed that allowed the expression of His-tagged biotinylated scFvs in Escherichia coli. The gene encoding a scFv specific for L. monocytogenes was cloned into pBAD380 and the His-tagged biotinylated anti-L. monocytogenes scFvs were coupled to streptavidin-coated IMBs. The ability of the anti-L. monocytogenes scFv-IMBs to capture Listeria monocytogenes and other Listeria species was evaluated in comparison to commercially available anti-Listeria IMBs. The anti-L. monocytogenes scFv-IMBs displayed higher efficiencies of capture (2.7%-17.4%) for strains of L. monocytogenes than was observed for the anti-Listeria IMBs (<1%-1.3%); also, the anti-L. monocytogenes scFv-IMBs exhibited improved specificity for L. monocytogenes as determined by cell capture efficiency in pure and mixed cultures.