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抗重组人血管生长素单克隆抗体的制备及鉴定 |
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杨太成 江悦华 冼 江 吴锦银 李 明
摘 要 目的：制备不同类型抗重组人血管生长素(Recombinantion Human Angiogenin,rhANG)的单克隆抗体，为进一步研究抗rhANG抗体对ANG的中和作用以及对ANG依赖型肿瘤的抑瘤实验打下基础。方法：利用杂交瘤技术以rhANG免疫Balb/c小鼠，制备二株(C6、C46)能够稳定分泌抗 rhANG抗体的杂交瘤，并对上清进行ELISA、双扩散、免疫印迹等鉴定。结果：该抗体类别C6为IgG1，C46为IgM，并特异地识别rhANG与其它细胞因子及血清蛋白无交叉反应，免疫印迹显示该抗体与分子量14.4 kD rhANG蛋白结合，不与宿主菌体蛋白反应。结论：经多种方法鉴定获得的二株杂交瘤细胞分泌的抗体为rhANG特异抗体。
关键词 单克隆抗体 rhANG 鉴定
中国图书分类号 R392.12
Preparation and identification of the anti-rhANG monoclonal antibodies
YANG Tai-Cheng,JIANG Yue-Hua,XIAN Jiang et al.
General Hospital of Guangzhou Military District,Guangzhou 510010
Abstract Objective: Different kinds of anti-human recombinant angiogenin(anti-rhANG)monoclonal antibodies were prepared in order to do further studies on the neutralization between ANG-dependant tumor.Methods:By applying hybridoma techniques,two cell lines(C6、C46)which can steadily secrete the anti-rhANG monoclonal antibodies were prepared from Balb/c mice immunized with rhANG、ELISA、two directions diffusion and Western blotting were applied to culture supernate identification.Results:One of the antibodies named as C6 is IgG1 and the other named as C46 is IgM.The antibodies could specifically recognized rhANG while had no crossing-reaction with other cytokines and serum proteins.The results of Western blotting showed that the antibodies could combine with rhANG protein whose MW is 14.4 kD,and had no reaction with host-thallus proteins.Conclusion:The antibodies secreted by two hybridoma cell lines identified by several methods were specific antibodies of rhANG.
Key words Monoclonal antibody rhANG Identification
血管生长素(ANG)是一种存在于正常血浆及实体瘤组织中的14.4 kD，PI 9.5的碱性多肽，是由肿瘤分泌，基因定位于染色体14q11［1］，它具有很强的促血管生长作用，大量的研究表明，ANG与恶性肿瘤的快速生成和转移，损伤组织的修复均有密切关系。本实验旨在制备不同亚型的抗人ANG单克隆抗体，研究ANG的拮抗剂对肿瘤血管的生成及肿瘤增殖、转移的抑制作用。
1 材料与方法
1.1 细胞因子 纯化rhANG，白细胞介素2(rhIL-2)由本室制备，rhGM-CSF，rhTNF-α及rhIFN-α国内产品。
1.2 抗体的制备[1] [2] [3] 下一页
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