Establishment And Preliminary Application Of Clostridium Chauvoei Dot-ELISA Method

Closridium chauvoei is a spore of Clostridium, which produces spores that can survive in the soil for a long term, invading the body through the digestive tract or mucous membranes, causing malignant edema and gas gangrene, they are infectious, that can infect humans. There is no standard serology preparation for the diagnosis of the disease. Therefore, this study aimed to hyperimmune serum antibody preparation for the establishment of a rapid, simple and sensitive serological diagnostic methods, laying a foundation for the development of the diagnostic reagent for gas gangrene in the future.This experiment successfully purified by effective antigen of Clostridium chauvoei standard strains C54-1 extraction, with highest specificity, purified antigen to immunize guinea pigs and serum obtained after four times immunizations to 1000 times dilution and by enzyme linked immunosorbent assay ELISA detecting the OD value of more than 1.6, and the Dot-ELISA method the repetitive experiments show that, the serum specificity and good stability, as Clostridium chauvoei double antibody sandwich Dot-ELISA for antibody in the detection.Screening the optimum reaction condition is the key point of the Dot-ELISA test. By repeated optimization, the best dilution of the antibody was determined to be 1:1024; the lowest cell antigen amount was 3.125 g, the minimum secretory antigen was 1.89 g, and the optimum enzyme labeled two was 1:4000. Using double antibody sandwich dot ELISA method, AGED and PCR methods simultaneous detection of 30 pathogenic in guinea pig liver and spleen milling liquid, the Dot-ELISA and PCR positive consistent rate was 93.3%, the positive detection rate is 2.5 times the AGED test method, the results that the method is highly sensitive. With the test set up the double antibody sandwich Dot-ELISA testing laboratory has confirmed the disease,30 positive samples were detected in 26 positive results, far higher than the detection rate of AGED method, the method is proved to have higher accuracy.This test is the first domestic application of standard strains of Clostridium chauvoei purified antigen to immune SPF guinea pig preparation of standard serum antibody coated membrane carrier, rapid detection of blackleg pathogen toxin double antibody sandwich dot ELISA diagnosis method is established. Compared with the method of ELISA method has higher sensitivity and specificity and can detect trace Clostridium chauvoei toxin and less amount of antibody, easy to be preserved, and does not require special equipment, convenient and quick operation, and is on track to produce kit was applied in clinical practice of blackleg rapid detection.