Study On Enzyme-Linked Immuno-Sorbent Assay Of Listeria Monocytogenes

Listeria monocytogenes (LM) is one of the most important foodborne pathogens, with the other three being E. coli O157, salmonella and Staphylococcus aureus. In all the serotypes of Listeria monocytogenes,4b type is the most common and cause adverse reactions of human and animal. The major symptoms caused by LM include Septicemia and Meningitis. In our study, an immunoassay had been developed for the rapid screening of LM in food samples. Our work includes three parts:(1) Preparation of flafellin of Listeria monocytogenesListeria monocytogenes (serotype 4b) being cultured at 22℃and 37℃, respectively, flagella were harvested by ultracentrifugation. The result was identified by SDS-PAGE and transmission electron microscopy. It showed that, L. monocytogenes produced a large number of flagella at 22℃, none or only one flagellum was produced at 37℃. In addition, high purity and high concentration (1.1mg/mL) of the flagellin was obtained with this method apart from time-saving.(2) Development of selective culture for LMIn this study, the effect of Staphylococcus aureus, Shigella, and E.coli O157 bacteria on LM was observed when FDA method was used. The results showed that selective media have some inhibitory effects on three kinds of bacteria. BLEB (adding Acridine flavin) is the best one, followed by BLEB without acridine flavin and TSB.(3) Development of LM Double-antibody Sandwich ELISAL. mocytogenes and flagellin will be used as antigen to obtain rabbit anti-LM polyclonal antibody and mouse anti-flagellin polyclonal antibody. Through five immunizations in ten weeks, antibody titers will be up to 106 or more. The concentration of the two purified antibody were 9.083 mg/mL and 7.940 mg/mL, respectively. The coating buffer, coating time, substrate action time, goat anti-mouse IgG:HRP dilution, and blocking liquid were studied to determine the optimization condition for double-antibody Sandwich ELISA for detection of L. mocytogenes, the conditions were as follows:coating buffer was 0.05 mol/L pH 9.6 Na2CO3-NaHCO3; substrate action time was 15 min; goat anti-mouse IgG:HRP was diluted by 5000 times;blocking liquid was 1％BSA；coating time was 37℃2h.