Monoclonal antibodies to DNA modified by a benzo[a]pyrene diol epoxide

Abstract

Monoclonal antibodies were obtained after fusion of mouse P3×63 Ag8.653 myeloma cells with spleen cells isolated from BALB/cCr mice immunized with either DNA modified by 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE-I-DNA) complexed electrostatically to methylated bovine serum albumin or with BPDE-I modified guanosine conjugated with bovine serum albumin, BPDE-I-G-BSA. One monoclonal hybridoma line from each type of immunization was grown as ascites tumors or in defined media and characterized in an enzyme linked immunosorbent assay (ELISA). The antibody produced from the spleen cells of a BPDE-I-DNA immunized mouse, designated 5D11, recognizes BPDE-I-DNA and DNA modified by 7β, 8α-dihydroxy-9β, 10β-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE II) but not unmodified DNA, N-2-acetylamino-fluorene (AAF) or 1-nitropyrene (NP) modified DNA, BPDE-II-dG or BPDE-I tetraol. It does recognize BPDE-I-dG but with a much lower affinity than when the adduct is present in DNA. In contrast, antibody 8E11 produced from the spleen cells of a BPDE-I-G-BSA immunized mouse recognizes the monoadduct BPDE-I-dG better than BPDE-I-DNA. It also recognizes BPDE-I tetraol but not BPDE-II-DNA, unmodified DNA, AAF- or NP-DNA or BPDE-II-dG. In a noncompetitive ELISA as little as 3 fmol of BPDE-I-DNA adduct can be detected with either antibody 5D11 or 8E11. The combination of the highly sensitive ELISA and highly specific monoclonal antibodies should be valuable in the detection and quantitation of human exposure to benzo-[a]pyrene.