Studies On Establishment Of Dot-ELISA For Detecting The Antibody Against Dirofilaria Immitis And The Antigenicity Of The Crude Soluble Proteins

Heartworm disease was a disease caused by Dirofilaria immitis which was a worldwide parasitic zoonosis, had an important effect on the development of animal husbandry-raising and human health. It had severely done harm to social economy and ecological environment.Being lack of special clinical symptoms, only the animals infected severely were easy to diagnose, but the animals without signs of visible clinical symptoms were difficult to find, so the early diagnosis of D.immitis infection was very difficult. Etiological detection was the main diagnostic tool of the D.immitis in our country at present. But the D.immitis -special serum antibodies had developed before the special signs of visible clinical symptoms presented, and a serological test using specific antigens had higher detection rate than etiological detection. So we developed Dot-ELISA to detect the specific antibody against D.immitis. In this study, the soluble proteins from D.immitis were extracted by freeze-thawed repeatly, ground and centrifuged. We have determined the best reacting conditions through repeatedly tests: the optimal coating concentration of antigen was 16.41μg/ml, the optimal coating condition was 37℃for 30min.1% BSA was the best blocking reagent, and blocked the antigens for 50min at 37℃. The working concentration of HRP-SPA was 1∶4000, serum sample for detecting and HRP-SPA should be incubated at 37℃for 1h, respectively. The sera dilution and wash buffer were both 0.01mol/L pH7.4 PBST(contain 0.15 mol/L NaC₁, 0.2% tween-20),the substrate for Dot-ELISA was incubated at 37℃for 15min. The high specificity of Dot-ELISA was proved by the specific blocking test, and also by the cross-reaction test in which the diaphragm did not react with the antibodies against Demodex canis, Ancylostoma caninum and Toxocara canis. The diaphragm had the ability to detect the positive serum when it was diluted to 1∶2⁸ and so it had good sensitivity; The repeatability test proved the method or the diagnostic diaphragm was stable. Stored at 4℃for at least 12 months, the diagnostic diaphragm's sensitivity and specificity didn't change, so it had good stability. Using Dot-ELISA to detect antibody can be finished in 3.5 hours and the result can be determined by naked eye without special instruments. All the results had shown that Dot-ELISA had many advantages. Firstly, it was a convenient, rapid, sensitive, specific method for detecting antibody. Besides, the diagnostic diaphragm is convenient to post or store because of it's stability. Fourthermore, all the materials and reagents can be easily standardized. Dot-ELISA provided a available technique for the detection and epidemiologic survey of D.immitis.In order to find the specific antigens for the diagnosis of D.immitis, the components of soluble antigens were firstly analyzed by SDS-PAGE technology, then these antigens were used to react with positive serum, sera from natural infection dogs and normal sera of dogs by using immunoblotting. The result of SDS-PAGE was that the soluble antigen revealed the complicated band pattern consisted of 23 protein bands. The major bands with molecular weight(MW) ranged from 19～120KD, The MW of major bands were 120,89,70,56,46,36,22 and 19kDa. The immunoblotting result showed that there were many bands recognised both by the positive serum and sera from natural infection rabbits, but in addition to those antigens, there were other antigens only recognised by the positive serum. In addition, there were differences in number of bands between sera from natural infection rabbits. The most number bands recognized was 10 separate bands, the least was 8 bands, but 120,70,56,36,22 and 19 kDa were both recognized by them.