Immunomagnetic Nanobeads For Highly Efficient Separation Of Listeria Monocytogenes

Listeria monocytogenes is an intracellular pathogenic bacterium causing a foodborne disease called listeriosis. This microbe can also grow over a wide range of temperatures (3-45℃), which makes it potentially hazardous, especially for refrigerated products. Listeria monocytogenes has tremendous harms which can seriously menace on human health as well as the economic development. It was important to establish a rapid, specific and sensitive method to screen LM in food samples. Generally complex sample processing and long enrichment or selective enrichment was required for pathogen detection to achieve detection sensitivity. Here we present a rapid and highly efficient method of sample preparation that enables specific separation of L. monocytogenes from food without an enrichment step.In the research, anti-LM polyclonal antibody was produced by Japanese rabbits using formalin inactivated LM cell debris as antigen. Then the antibody was used to couple magnetic beads after purified. The immunomagnetic beads (IMBs) were characterized by Laser particle size analyzer and transmission electron microscope. The capture efficiency of IMBs for Listeria monocytogenes with different coupling method (direct binding and streptavidin-biotin system) and different sizes (180nm,350nm and1150nm) were compared respectively. Many conditions, including the ratio of antibody to magnetic beads, the amount of IMBs, immunoreaction time and magnetic separation time were analyzed. At last, immunomangetic beads and traditional chromogenic medium were involved for evaluating the specificity and the capture efficiency of IMBs in food matrix.Results showed that the titer of purified anti-LM polyclonal antibody which had high binding affinity and specificity with LM was about640000. The capture efficiency of streptavidin-biotin immunomagnetic beads was about three-fold higher than that of directly binding ones. The capture efficiency of smaller beads is much higher than that of bigger ones. The key parameters were optimized:1mg magnetic beads added with100μg streptavidin,80μg antibody,0.1mg IMBs one time,45min for incubation time and3min for magnetic separation. The capture efficiency of IMBs can reach95%.The optimal coupling conditions and immunomagnetic beads capture process were achieved and the immunomagnetic beads showed good ability of enrichment. The streptavidin immuno-nano-magnetic beads possess three benefits of user-friendly format, a very short period for enrichment of Listeria monocytogenes and relatively inexpensive to make.