Anti-hBLyS Polyclonal, Monoclonal Antibody Preparation And Application

Human B lymphocyte stimulator (hBLyS) known in 1999 was a new member of the tumor necrosis factor (TNF) superfamily. It is closely related to the pathological mechanism of some immune diseases, and both low and over-expression of hBLyS can arise immune unbalance . In present study, Japanese big ear white rabbits and BALB/c mice were immunized with soluble hBLyS as antigen. Monoclonal antibody and polyclonal antibody were obtained, and some properties of them were identified respectively. Moreover, ELISA protocol has been established with the two kind of antibodies.1 Preparation and identification of anti-hBLyS polyclonal antibodyJapanese big ear white male rabbits (2~3 kg ) were injected with soluble hBLyS emulsified with Freund' s adjuvant every two weeks. The rabbits were bleed from the ear artery on the 11~th day since the third time immunization, and the titer of antisera was tested by indirect ELISA. Antisera titer reach to 1 : 5 × 10~5. Rabbits were killed, and blood was collected. Antisera was prepared and purifid with saturated ammonoium sulfate. The specificity of antiserum was determined with western-blot.2 Preparation and identification of anti-hBLyS monoclonal antibodyHBLyS was used as antigen to immunize BALB/c mice, and the splenocytes of immunized mice were fused with myeloma cells SP2/0. One hybridoma cell line secreting anti-hBLyS monoclonal antibody was obtained after the fusion cells and the subclone of 5 cycles. The numuber of the hybridoma chromatosome was between 87~101. The monoclonal antibody was IgG1, and the ascites titer can reach to 2×10~6. The western-blot result showed the specificity of the monoclonal antibody was excellent. The monoclonal antibody was produced by the method of inoculating BALB/c mice intraperitoneally with the hybridoma cells, and purified with protein A-Sepharose 4B affinity chromatography.3 Establishing of ELISA protocol to detect hBLySTwo kind of different ELISA protocols were tried to study the experiment conditionof detecting hBLyS level. Streptavidin was used to indirect coating in protocal 1, but biotin-avidin system was used to enlarge the final reaction signal in protocal 2. The optimal reaction condition was chosen by the experiments. The sensitivity of protocal 1 was poor, but the linear relation of protocal 2 standard curve was excellent when the concentration of hBLyS was between 3.2~400 ng/mL. So a protocal was established which sensitivity can reach to nanogram.