Antibodies to oxidative DNA damage: characterization of antibodies to 8-oxopurines

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 mumol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 mumol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 mumol/L. Both antibodies could easily detect the corresponding damage in x-irradiated f1 DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10,000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.