| Typhoid and paratyphoid are acute enteric communicable diseases transmitted by fecal-oral route, of which pathogenies are S. typhi and S. paratyphoid A B C. Typhoid and paratyphoid spread all over the world, approximately 16-17 millions peoples are infected by them every year, in which 0.6 million die, but most cases are in developing countries. More than 90% of the total incidence of typhoid and paratyphoid were typhoid previously, as the widely use of Vi bacterin, in the middle of 90s in 20th century, the popular strain changed, in some provinces the cases of typhoid decreased, but that of paratyphoid increased largely, with the outbreak trend from spot to part and from inshore provinces to backland provinces. In recent years, paratyphoid becomes the main infectious diease in some areas, and ofen develop to serious epidemic situation. So setting up a rapid and accurate detecting metod is necessary.The main methods of detecting and identifying S. paratyhpi A in lab are baterial culture, Widal agglutination reaction and typhiod hematocyte rapid diagnosis. However, these methods are time-consuming, labor-consuming, can't adapt to early diagosis, it's hard for epidemiologist to trace the source, earlily control, and for clicinian to diagnosis. The immunological methods built on enzyme-linked immunosorbent assay are specific, sensitive, simple and can detect vast samples simultaneously, have been used in clinical detecting pathogen already. But, by far there are rare studies about detecting salmonella parathphi A using ELISA. In our study, monoclonal antibody against 02 antigen of S. paratyphi A and polyclonal antibodies were prepared firstly, and then used to establish a sandwich indrect ELISA method.1. Preparation and identification of S. paratyphi A monoclonal antibodyS. paratyphi A contains three O-angigens: 01, 02 and 012, in which 02 antigen is unique. The bacterial body antigen was prepared by bathing in boiling water for 2h, and five female Balb/C mice were immunized. Two strains of hybridoma cell, 2C6 and 4F10, which could stably produce specific monoclonal antibody against 02 antigen were selected after 11 cell hybridization, of which ascites titer were 1:25600 and 1:12800 respectively. The molecular weight was both around 180 KD through SDS-PAGE analysis. The monoclonal antibodies produced by hybridoma cell, 2C6 and 4F10, didn't react with other strains in and out of salmonella species, so they were specfic to 02 antigen of S. paratyphi A.2. Establishment of sandwich indriect ELISA method The polyclonal antibodies were prodeced by immunizing the rabbits with the same antigen. The 2C6 monoclonal antibody, polyconal antibodies and HRP labeld goat anti mouse secondary antibody were chosen to build up a sandwich indrect ELISA method, detecting S. paratyphi A. The result indicated that the ELISA method was specific and there was no cross reaction with other baterias in and out of the salmonella species. The detecting limit could reach 10~5 CFU/mL, and the whole testing procedure only takes 4-5h. It offered a rapid, sensitive, accuate and efficient detecting method for the food detecting and clinical early diagnosis.Through the study above, the detection procedure of sandwich indrect ELISA was decided, and the reacting conditions were optimized. In future, the stablization and the use of the method also need to be tested, in order to set up the foundation to develop laboratory detection kits for S. paratyphi A.
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