Monoclonal antibodies and rabbit antisera recognizing 4-aminobiphenyl-DNA adducts and application to immunoaffinity chromatography

Monoclonal antibodies and rabbit antisera were produced that recognized 4-aminobiphenyl, its major DNA adducts and other metabolites. The antigens used to raise these antibodies were synthesized by coupling the aromatic amine to protein through a diazotization reaction. The goal of this immunization strategy was to induce antibodies that also cross-reacted with most 4-aminobiphenyl-derived metabolites. A total of 20 mice and four rabbits were immunized and every animal produced a strong immune response for 4-aininobiphenyl and its derivatives. Two IgG₁ monoclonal antibodies, 3D6 and 2E11, were isolated from two different mouse spleen cell fusions. One of the monoclonal antibodies, 3D6, had a high recognition for the three major 4-aminobiphenyl-DNA adducts: N-(deoxyguanosine-8-yl)-4-aminobiphenyl, N-(deoxyadenosine-8-yl)-4-aminobipbenyl and N-(deoxyguanosine-N²-yl)-4-aminobiphenyl, with affinity constants between 2 and 4 × 10⁹ l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 × 10⁹ l/mole. Thus, the strategy to use a diazotization coupling reaction was successful at producing high-affinity aminobipbenyl-DNA adduct-specific antibodies. Preparative immunoaffinity resins were made for each monoclonal antibody. These resins quantitatively bound 500 ng each [³H]N-acetyl-aminobiphenyl, [³H]N-aminobiphenyl and [³H]N-(deoxyguanosine-8-yl)-4-aminobiphenyl. Preliminary experiments were performed to test the applicability of the preparative monoclonal antibody immunoaffinity column to isolate [³H]4-aminobiphenyl-derived metabolites in dosed rat and dog urine. About 70% of the radioactivity in rat or dog urine could be bound to the immunoaffinity columns. The combined immunoaffinity column/HPLC analysis of the dog urine led to the identification of a novel urinary metabolite, N-formyl-aminobiphenyl. HPLC analysis of a rat urine sample tentatively found 4-aminobiphenyl, N-acetyl-4-aminobiphenyl and N-formyl-4-aminobiphenyl by co-chromatography, and these compounds accounted for 20, 6.8 and 6.5% of the total radioactivity in the chromatogram respectively. Taken together, these data show that these 4-amlnobiphenyl-speclfic monoclonal antibodies can be used in immunoaffinity columns to isolate metabolites and DNA adducts from biological samples.