Polyclonal Antibodies to DNA Modified with 4‐Nitroquinoline 1‐Oxide: Application for the Detection of 4‐Nitroquinoline 1‐Oxide‐DNA Adducts in vivo

Antibodies against 4-nitroquinoline 1-oxide (4NQO) adducts were elicited in rabbits immunized with 4NQO-modified DNA complexed with methylated bovine serum albumin. In enzymelinked immunosorbent assay (ELISA), the antibodies could recognize either denatured or native 4NQO-modified DNA, but not unmodified DNA, DNA modified with other carcinogens or free 4NQO derivative. Modification levels as low as 5 μmol of adduct per one mole DNA nucleotide (5 adducts/106 nucleotides) can be easily detected by the competitive ELISA. Indirect immunofluorescence staining by anti 4NQO-DNA antibody indicated that the antibodies bound specifically to the nuclei of normal human skin fibroblast cells treated with 4NQO. The intensity of fluorescence was proportional to the dose of 4NQO used to treat the cells, and the fluorescence positive cells could be detected after treatment with 0.25μM 4NQO (which resulted in the formation of 104 adducts per cell). Applying the competitive ELISA to the quantitation of DNA-adducts in rats treated with 4NQO, it was confirmed that the sensitivity of immunochemical assays was equivalent to that of isotopic assays. These methods should be helpful in studies on the formation of adducts and their removal in cells and tissues.