抗哇巴因多克隆抗体F(ab)_2片段的研制及鉴定

Preparation and identification of the F(ab)_2 fragment of rabbits polyclonal antibodies against human ouabain

目的 用胃蛋白酶酶解抗哇巴因多克隆抗体 ,制备出F(ab) 2 片段 ,为改型抗体的研究提供依据。方法 免疫家兔制备出抗哇巴因多克隆抗体 ,并用胃蛋白酶分别在不同的酶解条件下消化抗体 ,继之用高效液相排阻色谱法 (HPSEC)对其进行分析、纯化 ,采用酶联免疫吸附法(ELISA)检测、鉴定其活性。结果 用胃蛋白酶 2mg/1 0 0mg抗哇巴因多克隆抗体 ,消化 1 8h ,反应体系的 pH3 0时 ,可获得F(ab) 2 片段。结论 用胃蛋白酶酶解抗哇巴因多克隆抗体是研制出有活性的F(ab) 2

Objective Anti ouabain polyclonal antibody was digested with pepsin to prepare F(ab) 2 fragment in order to find the base of reshaped antibody. Methods Anti ouabain polyclonal antibody was prepared by immunizing rabbits. Then anti ouabain polyclonal antibody was digested under different conditions. The reactants were analyzed and purified by High Performance Size Exclude Chromatography (HPSEC). The immune activities were detected and identified by Enzyme Linked Immunosorbent Assay (ELISA). Results The proper reactive conditions for preparation of anti ouabain polyclonal antibody F(ab) 2 fragment with pepsin were established. The eligible dose of pepsin, by which 100mg anti onabain polyclonal antibody was digested better, was 2mg. The eligible digestive time was 18h. The value of pH of reactive system was 3.0. Conclusion The active F(ab) 2 fragment of anti ouabain polyclonal antibody can be obtained with pepsin. The method is simple and feasible.