ANTIBODIES TO OXIDATIVE DNA-DAMAGE - CHARACTERIZATION OF ANTIBODIES TO 8-OXOPURINES

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular pre mutagenic lesions produced in DNA by free radicals. Specific antibodie s were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihyd roadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produce d by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for th e haptens were elicited from rabbits immunized with the BSA conjugates . The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoG uo) precipitated the homologous antigens in an Ouchterlony gel diffusi on assay and no cross-reactivity was observed toward the normal nucleo sides or to the heterologous 8-oxopurine. Specificity was also examine d by hapten inhibition of antibody reactivity with the homologous conj ugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 mu mol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, ev en at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-ox oGuo for 8-oxoGuo was 0.1 mu mol/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 mu mol/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 i n irradiated DNA, and to standardize the ELISA signal, both products w ere measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled t o an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG r esidues 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.