Study On The Specificity Of Five Anti-echinococcus Monoclonal Antibodies

Echinococcosis,also known as hydatid disease,is an important zoonotic disease caused by the larvae of the genus Echinococcus genus;found throughout the world,most of hydatid disease which are caused by Echinococcus granulosus and Echinococcus multilocularis poses a great threat to the development of severe animal husbandry and human health worldwide.Echinococcus has a complex life history.Especially,there are many kinds of parasites in the intestinal tract of the terminal host dog.During immunodiagnosis,there will be crossover with other parasites,which brings certain difficulties to the prevention and control of hydatid disease.In this study,monoclonal ascites was prepared,salted out and purified to obtain monoclonal antibodies with high purity.The titer of monoclonal antibodies was detected by ELISA,and the purity of antibodies was verified by SDS-PAGE gel.Morphological identification was carried out with adults,soluble antigens were prepared,and anti-Echinococcus monoclonal antibodies were used for immunological analysis of the worm antigens,and the specificity of the five monoclonal antibodies was evaluated respectively result:(1)The titers of the five anti-Echinococcus monoclonal antibodies all reached more than 1:80 000.After SDS-PAGE electrophoresis,a light chain and a heavy chain could be clearly seen,which proved that the antibody purity was more than 90%,indicating that the preserved The five monoclonal cells still had a high ability to secrete antibodies after cryopreservation in liquid nitrogen.(2)Four kinds of middle-stage larvae were identified by morphology and molecular biology,and they were identified as: Echinococcus multilocularis,Echinococcus granulosus,Coenurus cerebralis,Coenurus cerebralis,and the monoclonal antibody against E.multilocularis protoscoleces was detected by ELISA.The larval monoclonal antibodies 1G11 and 1C1 did not cross-react with other worms,and specifically reacted with E.multilocularis protoscoleces;Western blot results showed that the monoclonal antibody1G11 could specifically recognize E.multilocularis protoscoleces antigen,and the recognition band was located at 66 ～ 270 k Da;m Ab 1C1 can specifically recognize E.multilocularis protoscoleces antigen,and the recognition band is between 80 ～ 270 k Da;both m Abs have no immunoblot reaction with the other three worms;immunohistochemistry It was shown that both monoclonal antibodies could recognize E.multilocularis protoscoleces antigen.(3)The six collected worms were identified based on their life history and morphology,and the six worms were identified as: E.multilocularis,E.granulosus,Taenia hydatigen,Taenia multiceps,Toxocara canis and Dipylidium caninum.Six helminth antigens were prepared,and their concentrations were detected by BCA kit method.Indirect ELSIA detection was performed on six helminth antigens,The results showed that monoclonal antibody 4D6 did not cross-react with other antigens,but only with E.multilocularis adult surface antigen.Western blot detection showed that the monoclonal antibody 4D6 could specifically recognize the surface antigen of E.multilocularis,and the recognition protein band was between 30 k Da and 120 k Da,and the monoclonal antibody 4D6 did not react with the other five parasite antigens in western blot.;Immunohistochemistry showed that the monoclonal antibody 4D6 could recognize E.multilocularis,and the reaction appeared on the surface of the worm.(4)The indirect ELSIA detection of six helminth antigens showed that the anti-E.granulosus adult surface antigen monoclonal antibodies 1F3 and 4E8 only reacted with the E.granulosus surface antigen,and the other five worms.The results of western blot showed that the monoclonal antibody 1F3 only recognized the E.granulosus surface membrane antigen,and the recognized antigen band was between 65 k Da and 170 k Da,while the monoclonal antibody 4E8 only recognized the E.granulosus surface membrane antigen,the recognition antigen band is 40 ～ 65 k Da;immunohistochemical results show that both monoclonal antibodies can react specifically with E.granulosus in brown color.(5)The five strains of cells have been tested above and have corresponding specificities,and they were all sent to the China Center for Type Culture Collection for preservation.The purpose of this study is to study the biological activity of anti-Echinococcus monoclonal antibodies,and further explore the antigenic epitopes recognized by each strain of monoclonal antibodies,which will lay the foundation for the development of subsequent immunodiagnostic techniques for parasites and the development of kits.