The Research Of Enrichment And ELISA Detection Methods For Salmonella

Salmonella is a kind of common foodborne pathogens in nature which caused the greatest number of food poisoning cases all around the world. The illness is characterized by acute diarrhea, vomiting, abdominal pain, fever and septicemia. It is a genus of Gram-negative, non-spore-forming and aerobic and anaerobic enteric bacilli which optimum growth temperature was37℃. Salmonella has complex antigenic structures and can be divided into three groups:somatic (O) antigens, flagellar (H) antigens and capsular (Vi) antigens. Since Salmonella 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 Salmonella in food samples. This research was developed IMS and double-antibody sandwich ELISA method for rapid detection of Salmonella in food samples.Female Japanese white rabbits were immunized with formalin inactivated Salmonella typhimurium and five mixed Salmonella bacteria to prepare polyclonal antibody. Caprylic acid ammonium sulphate precipitation and magnetic beads adsorption were used to purify polyclonal antibodies (pAb). The titer of the antibody was3.2×105. The pAb had serious cross reaction with other bacteria according to the result of dot blot hybridization. In this research, the antibody was purified by miscellaneous bacterial antigens in a reverse adsorption mode, which could successfully eliminate the cross reaction between the antibody and miscellaneous bacteria. Therefore, an effective purification method to obtain specific polyclonal antibody was fundamentally established.Anti-Salmonella typhimurium polyclonal antibody was coupled to the magnetic beads using EDC and NHSS method. The coupling conditions and immunomagnetic beads capture process were optimized. The results showed that the best technological parameters were:50μg antibody in lmg magnetic beads, the amount of immune-magnetic beads was0.3mg when105CFU/mL Salmonella was captured at one time, incubation time was45min and separation time was3min. The capture rates of the immunomagnetic beads for Salmonella in egg white, yolk, milk, and medium were44.3%,43.2%,65.2%and72.5%, respectively. The capture efficiency with Salmonella typhimurium. Salmonella choleraesuis, Salmonella parayohi A, Salmonella anatum and Salmonella enteritidis were61.4%,45.8%,48.2%,78.5%and64.1%when anti-mixed whole cell polyclonal antibody was used. In this study, anti-Salmonella immunomagnetic beads with high capture efficiency and specificity were made.In order to screen Salmonella in food samples, a rapid, highly specific and sensitive double-antibody sandwich ELISA was developed. In this method, the anti-Salmonella polyclonal antibody was used as capture antibody and C1360monoclonal antibody was used as detection antibody. It showed that Salmonella could be detected at a minimum of1.7×104CFU/mL in medium, and there was no cross reaction by using the sandwich ELISA method.