Monoclonal antibody to DNA containing thymine glycol

Abstract

Exposure of DNA to ionizing or near ultraviolet radiation modifies thymine to form ring-saturated products. One of the major products formed is 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol). Thymine glycol can also be selectively formed by oxidizing DNA with OsO 4. We have isolated hybrids that produce monoclonal antibodies against thymine glycol by fusing mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells from BALB/c mice immunized with OsO 4-oxidized poly(dT) complexed with methylated bovine serum albumin. This report describes the characterization of the antibody from one hybridoma using a competitive enzyme-linked immunosorbent assay (ELISA). The antibody reacted with both single- and double-stranded DNA treated with OsO 4, and with OsO 4-treated poly(dA-dT) and poly(dT); it did not crossreact with unmodified or apurinic DNA. It also reacted with DNA treated with H 2O 2 or with γ-rays at doses as low as 250 rad. We were able to detect 2 fmoles of thymine glycol in OsO 4-treated DNA and could quantitate 1 thymine glycol per 220 000 thymines. Using the antibody and the ELISA, the formation and removal of thymine glycol was examined in cultures of African green monkey cells irradiated with 25 krad of γ-rays. The antibody reactive sites produced by irradiation (8.5 per 10 6 thymines) were efficiently removed from the cellular DNA.