Miolo G, Stefanidis M, Santella R M, Dall'Acqua F, Gasparro F P
Department of Pharmaceutical Sciences, Padua University, Italy.
J Photochem Photobiol B. 1989 Feb;3(1):101-12. doi: 10.1016/1011-1344(89)80024-7.
The photochemical reactions of 6,4,4'-trimethylangelicin (TMA) with calf thymus DNA and an octanucleotide containing a single thymine have been characterized. HPLC analyses of enzymatically hydrolyzed TMA-DNA showed that isomeric forms of 4',5'-furan-side monoadducts were the major products. To develop monoclonal antibodies Balb c mice were immunized with the TMA-DNA complexed with methylated bovine serum albumin. The resultant antibodies were characterized by enzyme-linked immunosorbent assays (ELISA). The most sensitive antibody (7E3) has high specificity for TMA-DNA, very low cross-reactivity with DNA modified with either 4',5'-dimethylangelicin or 4'-methylangelicin and no cross-reactivity with non-modified DNA or with DNA modified with either 4'-aminomethyl-4,5',8-trimethylpsoralen or 8-methoxypsoralen. To characterize further this antibody, oligonucleotides containing specific TMA photoadducts were isolated from the photoreaction mixture by polyacrylamide gel electrophoresis and used as competitive inhibitors in the ELISA. Autoradiography of the gel showed an intense band corresponding to the 4',5'-monoadduct and two weaker unidentified bands. Antibody 7E3 reacted only with the 4',5'-monoadduct band as would be expected since this photoadduct was the principal photoadduct in the original antigen.
已对6,4,4'-三甲基白芷素（TMA）与小牛胸腺DNA以及含有单个胸腺嘧啶的八核苷酸的光化学反应进行了表征。对酶解后的TMA-DNA进行的HPLC分析表明，4',5'-呋喃侧单加合物的异构体形式是主要产物。为了制备单克隆抗体，用与甲基化牛血清白蛋白复合的TMA-DNA对Balb c小鼠进行免疫。通过酶联免疫吸附测定（ELISA）对所得抗体进行表征。最敏感的抗体（7E3）对TMA-DNA具有高特异性，与用4',5'-二甲基白芷素或4'-甲基白芷素修饰的DNA的交叉反应性非常低，与未修饰的DNA或用4'-氨甲基-4,5',8-三甲基补骨脂素或8-甲氧基补骨脂素修饰的DNA无交叉反应。为了进一步表征该抗体，通过聚丙烯酰胺凝胶电泳从光反应混合物中分离出含有特定TMA光加合物的寡核苷酸，并将其用作ELISA中的竞争性抑制剂。凝胶的放射自显影片显示出一条对应于4',5'-单加合物的强带和两条较弱的未鉴定带。正如预期的那样，抗体7E3仅与4',5'-单加合物带发生反应，因为这种光加合物是原始抗原中的主要光加合物。
