In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies
In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies
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DOI：
10.1016/j.dnarep.2004.05.016
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发表时间：
2004-11-02
期刊：
影响因子：
3.8
通讯作者：
Mori, T
中科院分区：
文献类型：
--
作者：
Iwamoto, TA;Kobayashi, N;Mori, T
本研究制备了能原位检测人细胞中N-乙酰氧基-2-乙酰氨基芴（NA-AAF）衍生的DNA加合物的单克隆抗体。作为免疫原，我们采用NA-AAF修饰的单链DNA静电耦合到甲基化的蛋白质，我们产生了五种不同的单克隆抗体。所有这些都显示出与NA-AAF修饰的DNA的强结合，但与未损伤的DNA的结合不可检测或最小。竞争性抑制实验显示，这些抗体识别的表位是DNA中的N-（脱氧鸟苷-8-基）-2-乙酰氨基芴（dG-C8-AAF），尽管DNA中的脱乙酰基N-（脱氧鸟苷-8-基）-2-氨基芴也以略低的效率被识别。相反，这些抗体不结合DNA中的3-（脱氧鸟苷-N-2-基）2-乙酰氨基芴或UV诱导的DNA损伤。有趣的是，它们仅显示出与小的AAF-核苷加合物（dG-C8-AAF）的最小结合，这表明除了加合物本身之外，DNA结合加合物侧翼的DNA区域对于抗体的稳定结合是必需的。用最有前途的抗体（AAF-1）进行酶联免疫吸附测定，我们检测了用生理浓度的NA-AAF处理的修复缺陷型着色性干皮病（XP）细胞的DNA中NA-AAF修饰的加合物的浓度依赖性诱导。此外，该测定能够证实正常人细胞有效地修复NA-AAF诱导的DNA加合物，而不是XP-A细胞。最重要的是，NA-AAF诱导的DNA加合物在XP细胞的单个核中的形成可以使用间接免疫荧光清楚地可视化。因此，我们成功地建立了新的单克隆抗体能够在原位检测NA-AAF诱导的DNA加合物在人类细胞。(C)2004 Elsevier B. V.保留所有权利。
The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derivedDNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N-2-yl)2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells. (C) 2004 Elsevier B.V. All rights reserved.