A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the afinity column. In studies on rats injected with ["'C]aflatoxin Bl, we identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFBl-N7-Gua), and the oxidative metabolites Ml and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins Ml and Pl. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.The aflatoxins are highly carcinogenic agents consistently found as contaminants in human food supplies in many areas of the world and epidemiologically linked to increased incidence of human liver cancer in Asia and Africa [see Busby and Wogan (ref. complex procedures used to purify the aflatoxins prior to analysis have seriously limited the application of these approaches in large-scale epidemiologic studies.A major objective of our work has been the development of rapid, noninvasive screening procedures for assessing the exposure of humans to environmentally occurring carcinogens. Useful protocols require the ability to quantify chemical carcinogens and their metabolites, especially DNA and protein adducts, in readily accessible compartments, such as urine and serum. We have been developing these monitoring techniques through immunoassays using monoclonal antibodies (8-10). These antibodies have proven to be useful analytical tools for quantifying the aflatoxins in biological fluids and also for use with affinity chromatography matrices as preparative tools to isolate aflatoxins from biological fluids (10). We report here results of initial applications of these techniques to the analysis of human urine obtained from people environmentally exposed to AFB1 in their diet and also of urine of rats injected with AFB1. These preparative and analytical procedures permit rapid measurement of aflatoxins in complex biological fluids under conditions that can be applied to large numbers of samples collected in epidemiologic surveys seeking to evaluate aflatoxin exposure as a risk factor for liver cancer in man.

Monoclonal antibodies specific for aflatoxin In a competitive radioimmunoassay using [3H]aflatoxin B1, 3pmol (1 ng) of aflatoxin B1, aflatoxin B2, or aflatoxin M1 caused 50% inhibition with this antibody. The antibody also had significant cross-reactivity for the major aflatoxin-DNA adducts: 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'oxo-N5-pyrimidyl)-3-hydroxyaflatoxin Bl. The antibody was also covalently bound to Sepharose-4B and used in a column-based solid-phase immunosorbent assay system. Aflatoxins added in vitro to phosphate buffer, human urine, human serum, or human milk at levels expected to be obtained in human samples acquired from environmentally exposed individuals were quantitatively recovered by applying the mixture to this antibody affinity column purification system. Preliminary studies using urine samples from rats injected with radiolabeled aflatoxin B1 have also indicated that aflatoxin metabolites can be isolated by these methods. Furthermore, we have found that the monoclonal antibody affinity columns can be regenerated for multiple use. Therefore, the monoclonal antibodies and their application to affinity chromatography represents a useful and rapid technique to purify environmentally occurring levels of this carcinogen and some of its metabolites for quantitative measurements.The aflatoxins are highly toxic and carcinogenic compounds, which are consistently found contaminating human food supplies in many areas of the world, and are epidemiologically linked to increased incidences of human liver cancer in Asia and Africa (1-3). Adverse biological consequences resulting from exposure to aflatoxin B1 (AFB1) are thought to be related to its ability to be oxidatively metabolized by constituative cellular enzymes. Of particular importance may be the ability of AFB1 to form covalently linked adducts with cellular macromolecules, including DNA. It is well established that AFB1 forms covalent DNA adducts following enzymatic oxidation to a highly reactive 2,3-epoxide, which nucleophil. ically attacks the N7 atom of guanine (4-6). The major DNA lesion produced by this reaction, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AF-N7-Gua), has been studied as an indicator compound of genotoxic damage to the cellular genetic material (7-9). A major objective of our work has been the development of noninvasive screening procedures for assessing the exposure of humans to environmentally occurring carcinogens. Useful protocols require an ability to quantify chemical carcinogens and their metabolites, especially DNA adducts, in readily accessible body fluids, such as serum and urine. We have been developing these screening methodologies by using immunoassays with monoclonal antibodies. We previously have reported production of monoclonal antibodies that recognize aflatoxin-DNA derivatives (10, 11). These early antibodies were found to be specific for AFBl-adducted DNA and in a competitive ELISA were able to detect AFB1 residues in DNA down to a...

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.