The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA

Antiserum was prepared in rabbits against ultraviolet-irradiated DNA. Using a ¹²⁵I-labeled protein A binding assay it was shown to be specific for ultraviolet-irradiated DNA, binding increasing as a function of logarithmic increase in dose. This antiserum was then used to develop a radioimmunoassay in which the competition between labeled UV-DNA and unlabeled sample DNA for antibody binding sites is monitored. Using this system the specificity of the assay could be changed depending on the nature of the labeled probe. The inability of poly(dA-dT) · poly(dA-dT), as compared with poly(dA) · poly(dT), to act as a competitive inhibitor established that the primary lesion recognized by the antiserum is the thymine dimer. This antigenic response did, however, depend on the presence of at least one nucleotide adjacent to the dimer. The sensitivity of the assay was optimized by using ³²P-labeled plasmid DNA as competitive probe and is capable of detecting photodamage in cellular DNA at doses as low as 2.5 J · m⁻².