Preparation Of Immunomagnetic Beads And Establishment Of Immunomagnetic Separation Method For Detection Of Listeria Monocytogenes

Objectives:To investigate the optimal conditions of magnetic beads coupled with antibodies and prepare the anti-listeria monocytogenes immunomagnetic beads. With combined use of immunomangetic beads and traditional chromogenic medium, a new method for separating and detecting listeria monocytogenes in food matrix has been initially established. The immunomagnetic separation had a promising prospect for inspecting the foodborne pathogenic bacteria.Methods:The cell of listeria monocytogenes was used as antigen for immunization of rabbit to prepare anti-listeria monocytogenes polyclonal antibody. The characteristics of polyclonal antibody including the binding affinity with listeria monocytogenes were determined. Laser particle size analyzer and transmission electron microscope were applied to analyze the particle size and dispersity of different groups modified magnetic beads. Immunomagnetic beads were prepared based on the stable magnetic beads. The optimum conditions of amine-modified magnetic beads coupling with antibodies were studied by orthogonal test and were selected to prepare the immnuomagnetic beads for capturing listeria monocytogenes. Three main options for carboxyl-modified mangnetic beads coupling with antibody were set:6 different coupling buffers, 6 main coupling time and 6 groups coupling temperature. The optimal coupling conditions were obtained by comparing the antibody surplus of supernatant after coupling. Carboxyl-modified immunomangetic beads and traditional chromogenic medium were involved for determining the capture capacity of newly prepared immunomagnetic beads to listeria monocytogenes.Results:The titer of purified anti- listeria monocytogenes polyclonal antibody which had high binding affinity with Listeria monocytoenes was about 1.3×105. The amine-modified magnetic beads of EM2-100/40 with an average size 743 nm and the carboxyl-modified magnetic beads of PM3-020 with an average of 180 nm were preferably dispersed. The optimal coupling condition for EM2-100/40 was pH9.6 and coupled for 1h at 37℃. The greatest amount of coupled antibodies was 68μg for 0.5 mg M2-100/40. The optimal working concentration of amine-modified immunomangetic beads was 50μg. With the coupling buffer of 0.01 mol/L 2-(N-Morpholino) ethanesulfonicacid(MES, pH6.0), purified polyclonal antibody was coupled with 1 mg carboxyl-Modified magnetic beads for 2 h at 37℃,and the amount of coupled antibody was 160μg. The capture ratio of newly produced carboxyl-modified Immunomagnetic beads was about 77%.Conclusion:The optimal conditions of coupled reaction were achieved and the Immunomagnetic beads were prepared successfully. Comparing with the traditional culture-based methods, the entire procedure for listeria monocytogenes detection can be reduced 20 hours at least with prepared immunomagnetic beads.