Preparation Of Monoclonal Antibodies And Fluorescent Beads Based Immnochromatographic Strip For Detection Of Listeria Monocytogenes

Listeria monocytogenes (L. monocytogenes) is a kind of food-borne pathogen, which can infect humans and other animals through contaminated food, drinking water and feces. This pathogen can grow well at4℃as well. Listeriosis could have a30%mortality rate, especially for pregnant women, newborns and immuno-compromised patients, the mortality rate would reach as high as70%. Therefore, the preparation of anti-Listeria-antibody with high specificity and affinity is one of the necessary conditions for establishing immunological detection methods of L. monocytogenes.In this study, the cell debris of L. monocytogenes was used as immunoantigen to generate L. monocytogenes specific monoclonal antibodies. Four monoclonal hybridoma cell lines named4A7,4H11,10A11and11E11, respectively, which can stably secrete monoclonal antibodies against L. monocytogenes, were obtained. Their antibody titers were1:160000,1:20000,1:160000,1:160000, and their antibody subclasses were IgG1, IgG2a, IgG1, IgG2a, respectively. Dot-ELISA assay showed that monoclonal antibodies from4A7,10A11and4E11harbor very well genus-specificity. Western-blot showed that monoclonal antibodies of4A7,10A11and4E11could bind with the antigen epitope of outer membrane proteins of L. monocytogenes, which molecular weights were62kDa,62kDa, and32kDa, respectively. The antibody of11E11could likely recognize the lipopolysaccharides of of L. monocytogenes, which molecular weight was in the range of14～24kDa, Colloidal gold immunoelectron microscopy was further used to indentify that the antibodies mentioned above could effectively recognize these antigen epitopes.We combined four anti-L. monocytogenes monoclonal antibodies (4A7、4H11、10A11、11E11) with two rabbit derived polycolonal antibodies(LM2, LM3) to develop the immunochromatographic assay. Results showed that4A7could couple with LM3very well, In the strip assay, LM3pAbs were used to lable the fluorescent beads,4A7-mAb and donkey-anti-mouse IgG were sprayed on nitrocellulose (NC) membranes as the test line and control line, respectively. The optimal experimental conditions of the fluorescent beads based immnochromatographic strip were as follow: the concentration of LM3was50μg per mg of fluorescent beads. The antibody4A7concentration of test line on NC membranes was0.5mg/mL, and the donkey-anti-mouse second antibody concentration of control line was1mg/mL. The volume of fluorescent bead and LM3conjugates (5mg/mL) was7μL for each of detection. Immunokenetic analysis of the strip demonstrated that the fluorescence intensity of both T line and C line kept increasing and would not reach a steady value within30min. However, the ratio of T/C could reach a stationary phase after20min of incubation. Under optimal conditions, the detection limits of this method was4.65×107CFU/mL, and the strips showed a high infinity to L. monocytogenes (containing17L. monocytogenes isolated from foods), Listeria ivanovii, Listeria welshimeri, Listeria seeligeri and Listeria innocua whereas exhibit no cross-reactivity with Listeria grayi and other30common bacterias.