Development Of Dot-ELISA And Colloidal Gold Strip Detection Methods To Echinococcus Granulosus Infection In Dogs

Canine Echinococcus disease is a kind of zoonotic parasitic diseases which is caused by Echinococcus granulosus parasites in intestinal of dogs. The disease is popular around the world, it gives a serious threat to human and animal health, blocks the development of animal husbandry. Canine is an important final host and also an important link during the epidemic of this disease. The detection methods of Echinococcosis are mainly divided into three categories, the traditional detection method, immunology detection method, and molecular biology detection method. Arecoline catharsis method of traditional detection method is the "gold standard" to detect the disease. However, this method has great limitation in clinical application because only 70% of dogs are sensitive to arecoline. Therefore, it is a key for prevention and treatment of Echinococcosis to find an effective, fast, convenient and not affected by outside conditions detection method. Antigen capture ELISA is very suitable for Echinococcus detection due to its strong specificity, high sensitivity, not affected by the level of individual animal immune response, convenient sampling, low requirement for the quality of the sample. This study established Dot-ELISA and colloidal gold strip with rapid、convenient and no special instruments based on three double-antibody sandwich ELISA methods established in our laboratory. This would provide an effective method and theoretical basis for the Echinococcosis prevention measures.First, this study purified monoclonal antibody 2D12 and polyclonal antibody of Ediag A864 antigen. Then the Dot-ELISA was developed with polyclonal antibody of Ediag A864 antigen which labeled by HRP and colloidal gold strip with monoclonal antibody of Ediag A864 antigen which labeled by gold particles detection methods to canine Echinococcus granulosus. This method aimed to provide new technology for the diagnostics of canine Echinococcus granulosus.Development of Dot-ELISA detection methods to Echinococcus granulosus infection in dogs This study established the Echinococcosis antigen capture Dot-ELISA detection method using monoclonal antibody 2D12 of Ediag A864 antigen and polyclonal antibody labeled with HRP. It got the best monoclonal antibody incubation concentration of 0.5μg/piece, the best sample dilution degree of 1:2, the best HRP-labeled antibody dilution degree of 1:400, the best blocking buffer of 5% skimmed milk, the best chromogenic time of 10 min. The application of Dot-ELISA detection method was used to detect 7 reference samples of Echinococcus granulosus infection in dogs gain from xinjiang province, the coincidence rate is 100% The sensitivity of Dot-ELISA detection method was 1:8. There was no cross reactivity with the positive fecal samples of Toxocara canis and Giardia canis, it got consistent test results using different batches of diaphragm.The application of Dot-ELISA detection method was used to detect 69 fecal samples(9 reference samples of Echinococcus granulosus infection in dogs gain from xinjiang province, 60 clinical samples gain from Changchun district), The detection rate of the 9 reference samples was 100%(9/9), the results of the 60 clinical samples were no posotive.Development of colloidal gold strip detection methods to Echinococcus granulosus infection in dogs This study established the Echinococcosis colloidal gold strip detection method using monoclonal antibody 2D12 of Ediag A864 antigen labeled with gold particles and polyclonal antibody of Ediag A864 antigen. The best dilution degree of gold-labeled antibody was 10:1, the best nitrocellulose membrane of Millipore 135 s, the best T line polyclonal antibody incubation concentration of 1mg/m L, the best C line antibody incubation concentration of 0.5 mg/m L. The application of Dot-ELISA detection method was used to detect 10 reference samples of Echinococcus granulosus infection in dogs gain from xinjiang province, the coincidence rate is 100%.The sensitivity of strip detection method was 1:4. There was no cross reactivity with the positive fecal samples of Toxocara canis and Giardia canis, it got consistent test results using different storage period(1 month, 2 months, 3 months, 4 months.)and stored condition( room temperature, 4℃, 37℃) of strips.The application of colloidal gold strip detection method was used to detect 96 fecal samples(36 reference samples of Echinococcus granulosus infection in dogs gain from xinjiang province, 60 clinical samples gain from Changchun district), The detection rate of the 36 reference samples was 97.2%(35/36), the results of the 60 clinical samples were no posotive.