血管生成素样蛋白2单克隆抗体的制备和分析

目的：研制抗人血管生成素样蛋白2（ANGPTL2）单克隆抗体,为进一步进行ANGPTL2的表达分析和功能研究,揭示其在糖尿病肾病发生发展过程中的可能作用创造条件.方法：以纯化的ANGPTL2重组蛋白皮下多点注射免疫BALB/c小鼠,分离脾细胞与小鼠骨髓瘤细胞融合,经HAT选择性培养基筛选,克隆和亚克隆化培养,ELISA鉴定,得到能稳定分泌抗人ANGPTL2的杂交瘤细胞;以硫酸铵沉淀法从杂交瘤细胞培养上清中初步纯化、制备抗人ANGPTL2单抗,以Western blot、细胞免疫染色和免疫组化染色相结合的方法对制备的单抗进行滴度和特异性鉴定.结果：得到1株能稳定分泌抗ANGPTL2的杂交瘤细胞,从其培养上清中制备的抗ANGPTL2单克隆抗体具有较好的特异性和较高的滴度,可用于Western blot、细胞免疫染色和免疫组化染色.结论：成功地研制了抗ANGPTL2单克隆抗体,为进一步进行ANGPTL2的表达分析和功能研究,揭示ANGPTL2的功能及其在糖尿病肾病发生发展过程中的可能作用创造了条件.

Preparation and characterization of monoclonal antibody against human angiopoietin-like protein 2

Objective:To prepare human angiopoietin like protein 2(ANGPTL2) monoclonal antibody.Methods:The purified recombinant human ANGPTL2 was used to immunize BALB/c mice.Then,the mouse spleen cells were isolated and fused with mouse myeloma cells.After selecting with HAT medium and analyzing with ELISA assay,the hybridoma cell clones stably secreting human ANGPTL2 antibody were screened out.The monoclonal antibody against humain ANGPTL2 was purified by ammonium sulfate precipitation method from the supernatant liquid of hybridoma cell culture.Western blotting,Cell immunostaining,and immunohistochemisty staining were used to characterize the antibody.Results:A strain of hybridoma cell clones stably secreting human ANGPTL2 antibody was screened out.The ANGPTL2 monoclonal antibody prepared was proven useful. Conclusion:A monoclonal antibody against human ANGPTL2 was successfully prepared,which provide a basis for basic study of ANGPLTL2.