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

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/thinlayer 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 concentration on single vs double binding by divalent antibody proteins. This technique is applied to photoproducts in whole inbred Xenopus laevis tadpoles, chronically irradiated at a series of UVB fluences that reach a lethality threshold when in vivo steady-state photoproduct levels are still quite low. As few as 0.01–0.02 CPDs per DNA kbp can be reliably detected, at signal/noise ratios of roughly 3∶1.