Induced Immune Tolerance For Therapy Of EAU

Objective: Since the uveitic pathogenic active of S-antigen is in relation to its purity, isolation of the antigen is extremely important. In this study, we simplified the production methods based on Al-Mahdawi's method, and acquired purified S-antigen rapidly and effectively. Immune Lewis rats with the S-antigen, establish EAU disease model and this afford a worthy tool for experimental study of immunogenic and therapy of uveitis.Methods: (1) Purification of S-antigen: S-antigen was purified by salt precipitation, molecular ethmoid and ion exchange chromatography. Retina extraction was prepared from freshly dissected bovine retina in ice. We selected Sephadex G 100 gel to exclude ammonium salt as well as molecular ethmoid. This method was simple and rapid and it has higher recycle rate. Then the sample was further isolated by DEAE-Sephadex A50 gel. Collect the effluent content S-antigen verified by SDS-PAGE electrophoresis, dialyze and concentrate, determine the protein content, adjust the content to 1mg/ml, keep frozen at -20℃ for use.(2) Analysis of S-antigen: ①biochemical analysis: Bradford method was used to measure the total protein content; calculate the molecular weight by compare with standard protein in inconsecutive SDS-PAGE electrophoresis. ② immunocompetence analysis: Emulsified S-antigen and CFA at 1:1 volume, inject 200μL the mixture in each hind foot pad, superadditional antigen was given from tail vein after a week, and constitute EAU animal model.(3) Establish EAU disease model: Female Lewis rats were selected between 6-8 weeks age(165-185g). Sufficient emulsified S-antigen and CFA at 1:1 volume,...