组织蛋白酶B酶联免疫吸附测定方法的建立

组织蛋白酶B（CB）与恶性肿瘤的分期、转移和预后关系密切。目的：建立CB酶联免疫吸附测定（ELISA）方法，以便评价肿瘤患者血清CB含量与预后的关系。方法：将含有CB cDNA的重组痘苗病毒转染Hela细胞，表达目的蛋白重组CB；经Western blot分析和制备型电泳纯化后，免疫新西兰大白兔，获得CB多克隆抗。纯体后的抗体一部分作为固化抗体，另一部分用辣根过氧化物酶标记；棋盘滴定法确定工作浓度。结果：痘苗病毒真核表达系统可以高效表达目的蛋白。制备型电泳能简单、有效地纯化蛋白。重组CB免疫动物后，可获得高效价的CB多克隆抗体，用以制备相应的固化抗体和酶标抗体检测CB。结论：用重组CB免疫动物后可获得相应抗体，以建立CB检测的ELISA方法。

Establishment of Enzyme-linked Immunosorbent Assay Method for Detection of Cathepsin B

Background: Cathepsin B (CB) highly corelates with the stage, metastasis and prognosis of malignant tumors. Aims: To establish an enzyme-linked immunosorbent assay (ELISA) method for detection of CB, and to evaluate the relationship between serum CB and the prognosis of cancer. Methods: Recombinant human CB was expressed by HeIa cells transfected by recombinant vaccinia virus. The expression of recombinant CB was detected by Western blot and purified by preparative electrophoretic systems. Then the New Zealand rabbits were immunized with purified CB in order to get its polyclonal antibodies. Parts of the antibodies were bound to solid phase, others were conjugated with horseradish peroxidase. The antibodies were diluted to determine its working concentration. Results: CB could be expressed in high yield by vaccinia virus eukaryotic expression system. By means of preparative electrophoretic systems, pure CB could be obtained. When purified CB was used to immunize the rabbits, high titer polyclonal antibodies could be gained. Two different kinds of antibodies could be used to establish a sandwich immunoassay to measure the concentration of CB. Conclusions: CB can be measured by ELISA method due to its specific antibodies.