Rapid Detection Of Listeria Monocytogenes Using Fluorescence Immunochromatographic Assay Combined With Immunomagnetic Bead Separation

Listeria monocytogenes is a zoonotic pathogenic bacterium causing a foodborne diseasecalled listeriosis. Listeria monocytogenes has tremendous harms which can seriously menaceon human health as well as the economic development. So it is extremely important to founda simple, rapid, sensitive and specific method to detect LM in food. In this study, a novelrapid method by combining immunomagnetic separation (IMS) and fluoresenceimmunochromatography assay (FICA) was developed for the detection of LM.Polyclonal antibodies (PAb) were produced by immunizing rabbit with the whole cells ofLM that were fixed by0.3%formaldehyde. The results of indirect ELISA showed that thetiter of antiserum was up to1/1.02×106. SDS-PAGE showed that by Protein G column affinitychromatography purification the purity of antibody was higher than bitterness-ammoniumsulfate precipitation method. The result of Western-blots indicated that the purified antibodyhad three specific junction belt with bacterial protein. The results of indirect ELISA showedthat purified antibody had good specificity and the titer was1/5.12×105, which was slightlylower than that of the beginning.Two kinds of magnetic beads which were micro and nanometer scale were used in theoptimization of coupling conditions. Then the capture efficiency of immunomagnetic beads(IMBs) and the specificity were evaluated. The results showed that the optimized parameterswere: activator EDC and sulfo-NHS, the reaction buffer pH6.0MES, coupling temperature37°C, coupling time2h,1mg magnetic beads added with80μg antibody，0.25mg IMBs onetime and45min for incubation time. The capture efficiency of nano immunomagnetic beadswas higher than micron-scale and the specificity of both IMBs were high in mixed bacteria.The antibody pairs were screened by fluoresence immunochromatographic platform. Theprocess conditions of immunochromatographic strip were optimized and the performance ofthe strips were also tested. Results showed that1mL fluorescent latex added with600μgantibody, antibody concentration of test line2mg/mL,24h for drying time of nitrocellulosemembrance, a100-fold dilution for latex, a1:2ratio for adding proportion and20min forreaction time. The detection limit of pure cultures was4×105CFU/mL. The average recoveries for immunochromatographic strips were in the range of96%-111%. The coefficientof variation of intra-assay and inter-assay was less than5%. The immunochromatographicstrips were quite good in stability and specificity. The detection limit of artificialcontamination samples was also4×105CFU/mL.In this paper, the elution efficiency of different elution buffer and particle sizes forimmunomagnetic beads were disscussed. The sensitivity, specificity and artificialcontamination detection of the established method was evaluated at the same time. Resultsshowed that the optimum elution method was10min of incubation at70°C in PBS buffer.The elution efficiency of micron immunomagnetic beads was higher than nano-scale. Thedetection limit of pure cultures samples was1×104CFU/mL when the sample wasconcentrated100times. The sensitivity of combined detection method was improved by afactor of40and the specificity was quite good. The detection limit of artificial contaminationsamples was also1×104CFU/mL. The detection sensitivity was not reduced compared withpure cultures.