Studies On Establishment Of Dot-ELISA For Detecting The Antibody Against Sarcoptes Scabiei Var.cuniculi And The Antigenicity Of The Crude Soluble Proteins

Scabies is a pruritic skin disease caused by Sarcoptes scabiei which is a worldwide parasitic zoonosis, has an important effect on the development of animal husbandry-raising and human health. It has severely done harm to social economy and ecological environment.Being lack of special clinical symptoms, only the animals infected severely are easy to diagnose, but the animals without scabies or signs of atopic dermatitis or other visible skin lesions are difficult to find, so the early diagnosis of mites infection is very difficult. Etiological detection is the main diagnostic tool of the sarcoptes scabiei in our country at present. But the scabies-special serum antibodies had developed before the special signs of atopic dermatitis presented, and a serological test using SS-specific antigens had higher detection rate than etiological detection. So we developed Dot-ELISA to detect the specific antibody against sarcoptes scabiei var.cuniculi. In this study, the soluble proteins from sarcoptes scabiei 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 12.03μg/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 1:40, 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 NaCl, 0.05% 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 RHD, Colibacillosis , Eperythrozoonosis and Mycosis. The diaphragm had the ability to detect the positive serum when it was diluted to 1:2¹⁰ and so it had good sensitivity; The repeatability test proved the method or the diagnostic diaphragm was stable. Stored at 4℃ for at least 5 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 2.5 hours and the...