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© 1992 Oxford University Press

research-article

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

John D. Groopman, Paul L. Skipper 1, Paul R. Donahue 1, Laura J. Trudel 1, Michael Wildschutte 1, Fred F. Kadlubar 2 and Steven R. Tannenbaum 1

Department of Environmental Health Sciences, The Johns Hopkins University, School of Hygiene and Public Health 615 North Wolfe Street, Baltimore MD 21205
1Division of Toxicology, Department of Chemistry, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge MA 02139
2Office of Research, National Center for Toxicological Research Jefferson AR 72079, USA

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 x 109 l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 x 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.


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