DNA adducts of nitropyrene detected by specific antibodies

Rabbit antisera containing polyclonal antibodies specific for the 1-nitropyrene derivatives, (1-acetylaminopyrene, 1-acetylamino-6-nitropyrene, 1-acetylamino-8-nitropyrene) and the major nitropyrene-DNA adduct, C-8-aminopyrene-deoxyguanosine, were obtained from New Zealand White male rabbits that were immunized with 1-nitrosopyrene-modified keyhole limpet hemocyanin (KLH). The affinity constants of the rabbit antisera for these derivatives ranged from 1 to 3 x 10(8) liters/mole. The ability of the antisera to detect 1-nitrosopyrene and the parent 1-nitropyrene was 25- to 30-fold less than the sensitivity to other metabolites. Female BALB/c and AJ mice were also immunized with 1-nitrosopyrene-modified KLH and 4 out of 18 surviving animals produced a low titer response when measured by an [3H] acetylaminopyrene-based radioimmunoassay. Mice that were immunized with a diazotized derived 1-aminopyrene bovine gamma globulin, 1-nitrosopyrene adducted bovine gamma globulin, and 1-nitrosopyrene-bound bovine serum albumin, produced very low immune responses. Spleen cells from selected mice were fused with myeloma cells but failed to produce stable clones that secreted nitropyrene-specific monoclonal antibodies. Therefore, the use of a 1-nitrosopyrene modified keyhole limpet hemocyanin to elicit an immune response specific for the nitropyrene moiety in one animal species (rabbit) was successful in producing a specific antisera. The immune response produced in mice and rabbits was much lower when compared to that produced by other chemically derived antigens we have used, such as the aflatoxins and 4-aminobiphenyl. The rabbit data encourages a continued attempt to produce monoclonal antibodies specific for nitropyrene. Such antibodies can be used in the development of preparative and analytical techniques to isolate and quantify nitropyrenes in biological samples from exposed human populations.