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目的:制备肝细胞胆管膜侧的单克隆抗体(mAb),并鉴定其特性。方法:利用密度梯度离心法提取大鼠肝细胞胆管侧膜小体,以测定特异性酶活性分离鉴定。分离成功的肝细胞胆管侧膜小体免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞常规融合制备抗体。间接ELISA法筛选抗体阳性的杂交瘤细胞并克隆化。采用免疫组织化学方法和Westernblot法对克隆化的mAb的抗原进行鉴定分析。结果:成功获得1株特异性分布于肝细胞胆管膜侧的mAb,命名为cm1。Western blot结果显示cm1Mr为140000;cm1在肝细胞胆管膜侧的膜小体内表达远远高于在肝细胞血窦侧膜小体内的表达。结论:成功地制备了肝细胞胆管膜侧特异性cm1mAb,并鉴定其作为极性分子分布于肝细胞胆管膜侧。
OBJECTIVE: To prepare a monoclonal antibody (mAb) on the hepatobiliary membrane and to characterize its properties. Methods: The bile duct of rat hepatocytes was extracted by density gradient centrifugation, and the specific enzyme activity was determined for isolation and identification. BALB / c mice were immunized with isolated hepatocellular bilaterally membrane-bound melanocytes. The spleen cells from the immunized mice were routinely fused with Sp2 / 0 myeloma cells to prepare antibodies. Antibody-positive hybridoma cells were screened by indirect ELISA and cloned. Antigen of cloned mAb was identified by immunohistochemistry and Western blot. RESULTS: One mAb specifically distributed on the membrane of hepatocyte bile duct was successfully obtained and named cm1. Western blot results showed that the cm1Mr was 140,000; the expression of cm1 in the intima of hepatocytes was much higher than that in hepatocytes. CONCLUSION: The hepatocyte bile ductial membrane-specific cm1 mAb was successfully prepared and identified as a polar molecule distributed on the hepatocyte bile duct membrane.