Fluoroimaging-Based Immunoassay of DNA Photoproducts in Ultraviolet-B-Irradiated Tadpoles

TL;DRAbstract

Sensitive and accurate measurement of photoproducts induced in DNA by natural or artificial ultraviolet-B (UVB; and UVC) light is essential to evaluate the toxic and mutagenic effects of this radiation. Monoclonal antibodies specific for the two major classes of photoproducts-cyclobutane pyrimidine dimers (CPDs) and pyrimidine-[6-4]-pyrimidinone photoproducts ([6-4]PPs)-have made possible highly specific and sensitive assays. Described here is the use of these primary antibodies with fluorescent secondary antibodies to generate 96-spot arrays. Stable fluorescence signals are rapidly and sensitively scored by fluoroimaging and computer analysis of peak-and-valley traces. CPD levels in a series of calibration standards are determined by acid hydrolysis/thin-layer chromatography analyses of radiolabeled bacterial DNA, UV-irradiated to known high fluences, and linear extrapolation to known lower fluences. The nonlinear fluorescence vs CPD curve reflects the effect of photoproduct concentra

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