شناسايي آنتي ژن اختصاصي كيست هيداتيك توسط روش وسترن بلاتينگ به منظور كاربرد تشخيصي

Identification of the specific antigen of hydatid cyst using western blotting method for diagnostic purposes

Background and Aim: Considering the importance of hydatidosis among human beings and domesticated animals, the aim of the present study was to identify the diagnostic antigen of hydatid cyst by immuno-blotting method. Material and Methods: After the preparation of sheep hydatid cyst, four kinds of antigens of raw fluids, boiled fluids, homogenized antigen of protosculex and the antigen of hydatid cyst wall were isolated. The negative and positive scrum samples without any impurity were prepared in slaughterhouse. Electrophoresis of antigens by means of SDS-PAGE method using disconnected gel along with isolating gel with the concentration of 12% and compressing gel with the concentration of 5% in the vicinity of a marker with certain molecular weight was carried out. In western blotting method for detection of the specific antigen, a sheet of nitrocellulose membrane was transferred over the gel and electrophoresis was performed. After transferring protein to nitrocellulose membrane, fatlcss powdered milk was used as a blocking buffer and then, primary and secondary antibodies and benzidin di-amino substrate were used accordingly to identify specific bands. Results: Electrophoresis pattern of the four antigens extracted from the cyst showed that among 14 protein bands, there are 9 protein bands with molccular weights of more than 116 Kilo-Dalton (kD) among which 4 bands with molecular weights of 18, 23, 40, and 64 kD were more prominent and similar to the antigens of boiled fluid. In the electrophoresis of wall antigen, 9 protein bands out of 14 bands had molecular weights of more than 116 kD and there were 10 protein bands with molecular weights of more than 35 kD in the protosculex. Conclusion: By immuno-blotting method in raw fluid antigen, a protein band with molecular weight of 35 kD, and in cyst wall antigen 3 protein bands with molccular weights of 30, 33, 46 kD were identified as specific antigens.