Groopman J D, Skipper P L, Donahue P R, Trudel L J, Wildschutte M, Kadlubar F F, Tannenbaum S R
Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Carcinogenesis. 1992 Jun;13(6):917-22. doi: 10.1093/carcin/13.6.917.
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.
制备了可识别4-氨基联苯、其主要DNA加合物及其他代谢产物的单克隆抗体和兔抗血清。用于制备这些抗体的抗原是通过重氮化反应将芳香胺与蛋白质偶联合成的。这种免疫策略的目标是诱导出能与大多数4-氨基联苯衍生代谢产物发生交叉反应的抗体。总共对20只小鼠和4只兔子进行了免疫，每只动物对4-氨基联苯及其衍生物都产生了强烈的免疫反应。从两次不同的小鼠脾细胞融合中分离出两种IgG1单克隆抗体，3D6和2E11。其中一种单克隆抗体3D6对三种主要的4-氨基联苯-DNA加合物：N-（脱氧鸟苷-8-基）-4-氨基联苯、N-（脱氧腺苷-8-基）-4-氨基联苯和N-（脱氧鸟苷-N2-基）-4-氨基联苯具有高识别性，亲和常数在2至4×10⁹ l/mol之间。此外，一种兔抗血清对DNA加合物的亲和常数为2.1×10⁹ l/mol。因此，使用重氮化偶联反应的策略成功制备出了高亲和力的氨基联苯-DNA加合物特异性抗体。为每种单克隆抗体制备了制备型免疫亲和树脂。这些树脂可定量结合500 ng的[³H]N-乙酰氨基联苯、[³H]N-氨基联苯和[³H]N-（脱氧鸟苷-8-基）-4-氨基联苯。进行了初步实验，以测试制备型单克隆抗体免疫亲和柱用于分离给药大鼠和犬尿液中[³H]4-氨基联苯衍生代谢产物的适用性。大鼠或犬尿液中约70%的放射性可与免疫亲和柱结合。对犬尿液进行免疫亲和柱/HPLC联合分析，鉴定出一种新的尿液代谢产物N-甲酰基-氨基联苯。对大鼠尿液样本进行HPLC分析，通过共色谱法初步发现了4-氨基联苯、N-乙酰基-4-氨基联苯和N-甲酰基-4-氨基联苯，这些化合物分别占色谱图中总放射性的20%、6.8%和6.5%。综上所述，这些数据表明这些4-氨基联苯特异性单克隆抗体可用于免疫亲和柱，从生物样品中分离代谢产物和DNA加合物。
