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-aminobiphenyl 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-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosine-N2-yl)-4-aminobiphenyl, with affinity constants between 2 and 4 x 10(9) l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 x 10(9) l/mole. Thus, the strategy to use a diazotization coupling reaction was successful at producing high-affinity aminobiphenyl-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-aminobiphenyl-specific monoclonal antibodies can be used in immunoaffinity columns to isolate metabolites and DNA adducts from biological samples.
Groopman
J D
JD
Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Skipper
P L
PL
Donahue
P R
PR
Trudel
L J
LJ
Wildschutte
M
M
Kadlubar
F F
FF
Tannenbaum
S R
SR
D000611:Aminobiphenyl Compounds / Q000032:analysis* / Q000276:immunology / Q000652:urine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002273:Carcinogens / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000276:immunology / Q000652:urine; D005260:Female; D007106:Immune Sera / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344
D000611:Aminobiphenyl Compounds; D000911:Antibodies, Monoclonal; D002273:Carcinogens; D007106:Immune Sera; C006757:4-biphenylamine; D004247:DNA