ANTIBODIES TO THE FOOD MUTAGENS, 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE AND 2-AMINO-3,4,8-TRIMETHYLIMIDAZO[4,5-F]QUINOXALINE - USEFUL FOR IMMUNOASSAY AND IMMUNOAFFINITY CHROMATOGRAPHY OF BIOLOGICAL SAMPLES

Monoclonal mouse IgG(1) and IgG(3) antibodies were developed to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) in order to make specific and sensitive detection and purification systems suitable for biological samples, The antibodies were developed with the strategy that cross-reaction with analogues modified in the N-2-position was desirable. Competitive enzyme-linked immunosorbent assays (ELISA) with 50% inhibition by 0.4-6 pmol food mutagen were developed, The epitopes recognized by the antibodies have been characterized by ELISA using 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx, and other food mutagens. One of the anti-PhIP antibodies only recognizes PhIP and those PhIP-analogues which have minor modifications in the N-2-amino group, whereas the other, 7B7-1, is less stringent and also recognizes several other modified metabolites, including bulky adducts at the Nz-amino group e.g. the major guanine and deoxyguanosine adducts isolated from PhIP-modified DNA, The antibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (4MeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8MeIQx), and the corresponding quinolines (4-MeIQ and 8-MeIQ), Two of these antibodies only bind analogues with minor modifications in the free amino group, whereas analogues with major modifications in this position, including a deoxyguanosine adduct, react with the third antibody. Urine samples and faecal extracts from H-3-PhIP or 2-C-14-DiMeIQx dosed rats were analysed by these ELISA assays, and high correlations between radioactivity and response in the ELISA assays were observed, Urine samples and faecal extracts from H-3-PhIP-dosed rats were purified on an affinity column containing the less stringent anti-PhIP antibody, 7B7-1, The affinity column was found by high performance liquid chromatography (HPLC) analysis to concentrate exclusively labelled material, This affinity column also bound PhIP-related materials from dilute samples of acid hydrolysed PhIP-DNA with high efficiency, Only similar to 40% of the 4,8-DiMeIQx related materials found in dilute acid hydrolysed samples of 4,8-DiMeIQx-DNA was bound by an affinity column containing the less stringent anti-4,8-DiMeIQx antibody, 2C5-1, We conclude that our anti-PhIP and anti-DiMeIQx antibodies can be used to determine the presence of these food mutagens and some of their activated or conjugated metabolites in complex biological samples.