Development Of Immunochromatographic Assays Based On Different Labels For Detection Of Salmonella Choleraesuis

Salmonella choleraesuis is an important main kind of foodborne pathogen. S.choleraesuis can cause gastroenteritis, typhoid fever, diarrhea, septicemia, and eventually death. The outbreak of S. choleraesuis is global. Human get poisoning by eating infected food. So, to develop efficient and rapid method to detect S.choleraesuis in food is a way to effectively cut off the route of transmission and prevent poisoning. In this study, three nanomaterials(fluorescent microspheres,magnetic nanoparticles, and gold magnetic bifunctional nanobeads) were researched and applied in sandwish immunochromatography assay.In chapter 2, an immunochromatographic test strip which based on fluorescent microspheres was developed. Antibodies amount and the pH of the system were optimized according to antibodies coupling rate and signal value of test line. Besides,the concentration of capture antibodies was optimized based on signal value of test line. The results showed as follows:the antibodies amount was 300 ?g/mg, the pH of the system was 6, and the concentration of capture antibodies was 2.0 mg/m L. The sensitivity of this method for detecting S. choleraesuis in pure culture was 1.9×106CFU/mL. There existed slightly cross reaction when detecting Staphylococcus aureus.In chapter 3, the immunomagnetic separation technique and fluorescent microspheres immunochromatographic assay were combined. The antibodies amount and the immunomagnetic nanoparticles amount were optimized based on capturing efficiency. In this assay the capture efficiency when detecting different concentration of S. choleraesuis was researched. Meanwhile, the time and temperature of heat treatment were optimized. The results were that the antibodies amount was 30 ?g/mg,the immunomagnetic nanoparticles amount was 100 ?g/mL, the capture efficiency when detecting different concentration of S. choleraesuis can reach 90%, and the time and temperature of heat treatment were 10 min and 75 ?. The sensitivity of the method that combined the immunomagnetic separation technique with fluorescent microspheres immunochromatographic assay was 1.52×105 CFU/mL. The sensitivitywas enhanced 10 times compared with that of fluorescent microspheres immunochromatographic assay.In chapter 4, a novel immunochromatographic test strip with gold magnetic bifunctional nanobeads was developed. At first, the gold magnetic bifunctional nanobeads were characterized. Then, the pH of the system, antibodies amount and the immune gold magnetic bifunctional nanobeads amount were optimized according to capture efficiency. Besides, the specificity of immune gold magnetic bifunctional nanobeads and the detection of S. choleraesuis in different concentration was tested.In the procedure of conjugation between GMBN and anti-S. choleraesuis antibodies,the pH of system was 6 and 150 ?g/mg of antibody was added. Twenty microgram of the antibody-GMBN complex was used to capture 1 mL of S. choleraesuis. The capture efficiency when detecting different concentration of S. choleraesuis was more than 80%. The S. choleraesuis-antibody-GMBN complex could be used to detect S.choleraesuis in immunochromatographic test strip. Results showed that the sensitivity of the strip was 5×105CFU/mL which was enhanced 10 times compared with gold immunochromatography assay. Besides, the immunochromatographic test strip could be applied in food samples(whole milk) and the entire detection time was 20 h.