USE OF THE SINGLE-CELL GEL-ELECTROPHORESIS ASSAY FOR THE IMMUNOFLUORESCENT DETECTION OF SPECIFIC DNA-DAMAGE

The single-cell gel electrophoresis assay or comet assay is now a wide ly used method to assess the level of DNA damage in irradiated or chem ically modified cells. We propose an adaptation of the currently appli ed protocol, aimed at singling out a defined modified base, using an i mmunodetection approach. After the electrophoresis step, the DNA tail moment was measured using ethidium bromide. Simultaneously, cyclobutan e pyrimidine dimers (CPDs), the targeted lesions, were revealed by an indirect immunofluorescence detection using a specific monoclonal anti body. The assay was validated on human fibroblasts exposed to UVB ligh t. The dose-response curves were established, showing a linear increas e of the antibody response with the dose between 1000 and 10,000 J/m(2 ). The detection limit of the method was 500 J/m(2). Digestion of the CPDs, induced at 3000 J/m(2), with T4 endonuclease V led to a marked d ecrease of the antibody response, confirming the specificity of the as say. A preliminary repair experiment is reported in which the tail mom ent of the comets together with the antibody response are measured, sh owing the disappearance of 80% of the antibody fixation sites within 4 8 h. (C) 1998 Academic Press.