A SENSITIVE IMMUNOASSAY TECHNIQUE FOR THYMINE DIMER QUANTITATION IN UV-IRRADIATED DNA

This paper describes an immunological technique for thymine dimer quan titation which is more sensitive than the high performance liquid chro matography (HPLC) method used previously in our laboratory. Calf thymu s DNA was irradiated in the presence of the photosensitizer acetopheno ne to induce the formation of cis-syn thymine dimers exclusively. Thes e DNA solutions were then denatured and injected into New Zealand whit e rabbits to raise antibodies against the thymine dimer. Blood was dra wn from the rabbits at regular intervals and the crude serum was used in an immunoblotting protocol which immobilized the antigen-antibody c omplex on a membrane system. Subsequent detection and quantitation of the thymine dimer antigen was performed by enhanced chemiluminescence (ECL). Dilutions of the antibody used in the above protocol could be q uantitatively related to the UV-irradiated DNA antigen, i.e. the thymi ne dimer. It is shown that this technique is 4000-8000 times more sens itive than the HPLC technique used previously. The above immunoblottin g/ECL protocol was then used to test the validity of a proposed mechan ism for acetophenone photosensitized dimerization of thymine in DNA at concentrations more relevant to cellular systems, but previously unde tectable by HPLC.