| Objective: To approach the potential of the protein of Schistosomulum japonicum i nthe diagnosis of schistosomiasis. We Preparation of excreted a nd metabolic antigensfrom adult worm of Schistosoma japonicum， to analyze thecomponetns、.immunogenicity and immunoreactivity. Meanwhile, Mice wereimmunized with the antigens,we generate and identify monoclonal antibodies againstSj-EMAs.to analyze the antibody subtype、 the positioning of the adlut andwestern-bloting for evaluation its potential immunodiagnostic value of detectingcirculating antigen in the patients with schistosomiasis. Methods: Rabbits wereinfected with cercariae(about2000/rabbit) and killed after42days for obtaining adultworms of Schistosoma japonicum. Adult worms were incubated in sterilized serum-freeDMEM medium at37℃for4h．The supernatant was dialysed against PBS and theSj-EMAs were obtained. Mice were immunized with the antigens. The serum antibodytiter was determined by ELISA；polystyrene microtitre plates were coated with thisantigens，and antibody titer of individuals infected with S.japonicum was determined；SDS-PAGE was carried out to analyze Sj-EMAs components； Then Western blot wascarried out to screen the protein that was able to react with the anti-Sj-EMAsmonoclonal antibody(Hybridoma supernatant stored in this room)．Simultaneously, afterthe6week-old female BALB/c mice were immunized three times with Sj-EMAs，thespleen cells of the immunized mouse were fused with myeloma cell line SP2/0，Thehybridoma cells were selected in the HAT selective medium, By subcloning andexpansion of training, we have prepared hybridoma cells that can stably secretinganti-Sj-EMAs monoclonal antibody. Preparation of monoclonal antibody ascites byBALB/c female mice,this ascittes were purificated by protein G affinitychromatography after Crude purification of saturated ammonium sulfate. the Ig subtypes of the McAb were assayed with the Sigma Mouse Monoclonal AntibodyIsotyping Reagents Kits., Indirect immunofluorescence assay to observe the recognitionsites of two monoclonal antibodies in th e adult S. japonicum, Western-Blottingexperiments identified Immunological charact eristics of the two hybridoma cell lines.Results: Sj-EMAs were successfully prepared；the antibody titer of immunized micewas1:16000；the antibody titer of patients with acute schistosomiasis，chronicschistosomiasis patients，patients with advanced schistosomiasis and hookworm are1：800，1：400，1：800and1：100，respectively；There were4common protein bandsin the SDA-PAGE analysis，with the molecular weights of13，40，60and180kd，respectively；A positive protein band can be identified by anti-Sj-EMAs monoclonalantibody，with a molecular weight of5kd．Two hybridoma cell lines that can stablysecreting monoclonal antibodies against Sj-EMAs were harvested and identified,after invitro repeatedly passaged, both of the hybridoma cell lines still could secrete high titerantibodies,and Purified ascites also had a higher titer. The two McAb Isotypes bothbelongs to IgG1, Immunofluorescence experimental results showed that the recognitionsites of the two monoclonal antibodies are in the membrane of adult S. japonicum.Western-Blotting confirmed that the two cells are secreting anti-Sj-the EMAsmonoclonal antibody, A positive protein band of Sj-EMAs can be identified byanti-Sj-EMAs monoclonal antibody，with a molecular weight of25kd. Conclusion:This research has obtained excreted and metabolic antigens from adult worm ofSchistosoma japonicum, It confirmed that Sj-EMAs have strong immunogenicity andimmunoreactivity. At the same time,Two hybridoma cell lines that can specificysecreting monoclonal antibodies against Sj-EMAs were harvested and identified, he twoMcAb Isotypes both belongs to IgG1, Western-Blotting results confirm theimmunological characteristics of the two monoclonal antibodies, Immunofluorescenceassay results confirmed that the recognition sites of the two monoclonal antibodies arein the membrane of adult S. japonicum. |
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