抗单链DNA单克隆抗体的制备及应用

目的:制备特异性的抗单链单克隆抗体,为建立一种特异、敏感、简便的细胞凋亡检测方法奠定基础。DNA方法:根据烷化剂盐酸氮芥能特异性地修饰小牛胸腺链上的鸟嘌呤而暴露互补链上的胞嘧啶这一特性,将修饰后的DNA(G)(C)小牛胸腺连于甲基化的牛血清白蛋白上制备成完全抗原,免疫小鼠获得特异性的抗单链单克隆抗体,并对DNABALB/cDNA单抗的类别和特异性进行鉴定;用叠氮钠和抗肿瘤药物诱导了细胞的坏死和凋亡,利用所制备的抗单链单克VP-16HL60DNA隆抗体对其进行检测,并与形态学方法、凝胶电泳法和法进行了比较。TUNEL结果:所制备的抗单链单克隆抗体能与羊DNA抗小鼠发生特异性反应,且能与凋亡细胞特异性结合,而不结合坏死细胞。IgM结论:所制备的单抗为型,利用其能特异IgM地结合单链的特性,可用于检测细胞凋亡,简便、有效地区别细胞凋亡和坏死。DNA

Preparation of monoclonal antibody against single-stranded DNA

Objective: To prepare the monoclonal antibody (McAb) against single-stranded DNA(ssDNA) and examine apoptotic cells. Methods: Based on the exposure of the deoxycytidines(C) on the opposite DNA strand by guanines (G) selective alkylation treated with nitrogen mustard(HN 2), the calf thymus DNA treated with HN2 and conjugated with methylated bovine serum albumin(MBSA) was used to immunize BALB/c mice and the monoclonal antibody (McAb) against single-stranded DNA(ssDNA) was prepared.HL60 cell apoptosis was induced by treatment with etoposide(VP-16) and cell necrosis was induced by exposure to sodium azide(NaN 3). Apoptotic and necrotic cells were examined by anti-ssDNA McAb stain ,TUNEL assay and agarose gels electrophoresis. Results: Anti-ssDNA McAb can react with goat-anti-mouse IgM and conjugate apoptotic cells. Conclusion: The anti-ssDNA McAb is IgM antibody and can distinguish the apoptotic cells and the necrotic cells.