Preparation and characterization of antibodies against 3,2'-dimethyl-4-aminobiphenyl-modified DNA

Abstract

Polyclonal antibodies for 3,2'-dimethyl-4-aminobiphenyl (DMAB)-DNA adducts were obtained from the sera of rabbits immunized with N-OH-DMAB-modified DNA. Using the enzyme linked immunosorbent assay (ELISA), these antibodies were shown to recognize DNA modified by N-OH-DMAB and N-hydroxy-4-aminobiphenyl, but not unmodified DNA, 2-acetylaminofluorene- or 4-nitroquinoline-1-oxide-modified DNA, free aminobiphenyl or DMAB derivatives. Using competitive ELISA with DMAB-modified denatured DNA, a 50% inhibition of antibody antigen binding was caused by 7 fmol of DMAB adducts applied to each assay well as an inhibitor. Native DNA bearing adducts was associated with 50% inhibition in the range of 22-90 fmol/asay well, depending on the levels of modification. The adducts recognized by the antibody were shown to be stable against treatment of heat or alkali denaturation. Optimal conditions for the detection of adducts in DNA from the rat organs exposed to DMAB were established by means of competitive ELISA, using alkaline-denatured DNA as an inhibitor. Thus, as little as 5 fmol adducts in 1 microgram of DNA could be detected. Indirect immunofluorescence staining indicated that anti-DMAB-DNA antibodies bound specifically to nuclear components of rat fibroblast 3Y1 cells treated with N-OH-DMAB. The intensity of the fluorescence was proportional to the dose of carcinogen administered. The antibodies should be helpful for use in studies on the formation of adducts and their removal in cells and tissues after DMAB administration.