| Dirofilariasis, also known as Heartworm disease, is mainly caused by the Dirofilaria immitis that mainly parasite in canine right ventricular and pulmonary arteries. The parasite cause severe disease and even death in dogs, cats and other animals in many parts of the world. Microfilaria, the larver of Dirofilaria immitis that can be found in the blood of infected animals, are usually spread by mosquitoes. Sometimes, it may even infected human beings, leading to pulmonary nodules. The dirofilariasis is widely distributed in China. The epidemiological investigation of some areas showed that the infection rate was more than 40%. However, there is a lack of rapid and convenient method for clinical detection. Therefore, it is very important to establish a specific, sensitive and efficient immunological diagnosis method for the diagnosis and prevention of the disease.The monoclonal anti-MTFP antibody has been developed with specific MTFP protein which was obtained by screening c DNA library in our lab. And then the localization of MTFP protein antigen in Dirofilaria immitis was determined. The Dot-ELISA has been established using the purified MTFP recombinant protein as coating antigen. And then colloidal gold strip was developed with MTFP recombinant protein as the T line antigen. In the end, a sample detection method for the diagnosis of infection of Dirofilaria immitis in dogs is established. 1. Preparation and purification of Anti-MTFP monoclonial antibodyIn this study, Balb/c mice were immunized with MTFP recombination protein. Then, the spleen cells of immunized mice were fused with SP2/0 myeloma cells and the cell fusions, 5 strains of the positive hybridoma cells were selected and named No 7,8,11,16,20, respectively. After identification of antibody subtype, number 11 was Ig G1 subtype, number 8 and 16 were Ig G2 a subtype, number 2 and 20 were Ig M subtype. No.16 strain was purified by affinity chromatography. The efficiency of purification was perfect. ELISA diction showed the antibody titer of number 16 was 2×105. 2. The localization of MTFP in Dirofilaria immitisWith Anti-MTFP monoclonial antibody, No 16, MTFP was localized by laser scanning confocal microscopy. Finally, MTFP gene was found to be expressed in muscle layer of adult worm body. 3. The establishment of Dot-ELISA detection method of Dirofilaria immitisIn this study, Dot-ELISA detection method of Dirofilaria immitis was established with Dirofilaria immitis MTFP recombination protein. Through the optimization of reaction conditions, the best recombination protein incubation concentration was 0.5μg/piece, the best sample dilution was 1:50, the best HRP-labeled antibody dilution was 1:4000. The sensitivity of Dot-ELISA detection method was 1:200. There was no cross reactivity with the positive serum samples of Isospora rivolta and Giardia canis. The method was used to detect 62 canine serume samples from Baicheng city, Jilin Province. The result showed that three samples were positive, the positive rate was 4.8%. 4. The establishment of colloidal gold strip detection method of Dirofilaria immitisIn this study, colloidal gold strip detection method of Dirofilaria immitis was established with Dirofilaria immitis recombination protein. The best nitrocellulose membrane was Millipore 135 s, the best material for gold standard pad was glass fiber RB45, the best material for the sample pad was glass fiber SB08, the best T line incubation concentration was 1mg/m L, the best C line antibody incubation concentration was 0.5 mg/m L. The sensitivity of strip detection method was 1:8. There was no cross reactivity with the positive serum samples of Toxocara canis and Isospora rivolta. The stability test showed that the strips could be stored at RT for at least 2 months and the repeatability was good. The strips were used to detect the 62 samples that had been detected by the Dot-ELISA detection method of Dirofilaria immitis. The result showed that 4 samples were positive, the positive rate was 6.4%. In conclusion, a simple detection method for Dirofilaria immitis infection is established. |
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