A sensitive immunoassay technique for thymine dimer quantitation in UV-irradiated DNA

Abstract

This paper describes an immunological technique for thymine dimer quantitation which is more sensitive than the high performance liquid chromatography (HPLC) method used previously in our laboratory. Calf thymus DNA was irradiated in the presence of the photosensitizer acetophenone to induce the formation of cis-syn thymine dimers exclusively. These DNA solutions were then denatured and injected into New Zealand white rabbits to raise antibodies against the thymine dimer. Blood was drawn from the rabbits at regular intervals and the crude serum was used in an immunoblotting protocol which immobilized the antigen-antibody complex 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 quantitatively related to the UV-irradiated DNA antigen, i.e. the thymine dimer. It is shown that this technique is 4000–8000 times more sensitive than the HPLC technique used previously. The above immunoblotting/ECL protocol was then used to test the validity of a proposed mechanism for acetophenone-photosensitized dimerization of thymine in DNA at concentrations more relevant to cellular systems, but previously undetectable by HPLC.