Sauvaigo S, Serres C, Signorini N, Emonet N, Richard M J, Cadet J
Département de Recherche Fondamentale sur la Matière Condensée, SCIB/LAN, CEA Grenoble, France.
Anal Biochem. 1998 May 15;259(1):1-7. doi: 10.1006/abio.1998.2628.
The single-cell gel electrophoresis assay or comet assay is now a widely used method to assess the level of DNA damage in irradiated or chemically modified cells. We propose an adaptation of the currently applied protocol, aimed at singling out a defined modified base, using an immunodetection approach. After the electrophoresis step, the DNA tail moment was measured using ethidium bromide. Simultaneously, cyclobutane pyrimidine dimers (CPDs), the targeted lesions, were revealed by an indirect immunofluorescence detection using a specific monoclonal antibody. The assay was validated on human fibroblasts exposed to UVB light. The dose-response curves were established, showing a linear increase of the antibody response with the dose between 1000 and 10,000 J/m2. The detection limit of the method was 500 J/m2. Digestion of the CPDs, induced at 3000 J/m2, with T4 endonuclease V led to a marked decrease of the antibody response, confirming the specificity of the assay. A preliminary repair experiment is reported in which the tail moment of the comets together with the antibody response are measured, showing the disappearance of 80% of the antibody fixation sites within 48 h.
单细胞凝胶电泳试验或彗星试验是目前广泛用于评估受辐照或化学修饰细胞中DNA损伤水平的方法。我们提出了一种对当前应用方案的改进方法，旨在使用免疫检测方法分离出特定的修饰碱基。电泳步骤完成后，使用溴化乙锭测量DNA尾矩。同时，通过使用特异性单克隆抗体的间接免疫荧光检测来揭示作为目标损伤的环丁烷嘧啶二聚体（CPD）。该试验在暴露于UVB光的人成纤维细胞上得到验证。建立了剂量反应曲线，结果表明在1000至10,000 J/m²之间，抗体反应随剂量呈线性增加。该方法的检测限为500 J/m²。用T4内切核酸酶V消化在3000 J/m²诱导产生的CPD，导致抗体反应显著降低，证实了该试验的特异性。报告了一项初步修复实验，其中测量了彗星的尾矩以及抗体反应，结果显示在48小时内80%的抗体固定位点消失。
