POLYCLONAL ANTIBODIES TO ADENINE N-1-OXIDE - CHARACTERIZATION AND USEFOR THE MEASUREMENT OF DNA-DAMAGE

Adenine N-1-oxide is a DNA lesion whose formation involves the specifi c oxidation of the adenine base by hydrogen peroxide under nonradical conditions. The damage may be measured using a HPLC/P-32-postlabeling method, which however cannot be used for routine analysis. We propose herein as an alternative an immunological assay which allows a rapid e valuation of the level of adenine N-1-oxide in DNA exposed to oxidativ e stress. Two polyclonal antibodies were raised using two different st rategies for the coupling of the hapten to the protein. The first appr oach is based on the universal method of Erlanger and Beiser, whereas the preparation of the second antigen involves the conjugation of a mo rpholino derivative of adenosine N-1-oxide to the carrier protein. The affinity and the specificity of those antibodies were determined by c ompetitive enzyme-linked immunosorbent assay. The antibody obtained by the traditional method shows some cross-reactivity with normal nucleo tides, whereas for the other antiserum the selectivity was found to be higher. Therefore, this polyclonal antibody was used to quantify the level of adenine N-1-oxide in calf thymus DNA oxidized either by m-chl oroperbenzoic acid or by hydrogen peroxide. The detection limit of the assay is four residues of adenine N-1-oxide per 10(6) normal bases. T he level of adenine N-1-oxide in nonmodified DNA was lower than the de tection limit of the assay, whereas in mCPB- and H2O2-modified DNA, it could be up to 14 and 0.7 adenine N-1-oxide residues per 10(4) normal bases, respectively.