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

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Monoclonal mouse IgG1 and IgG3 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 N2-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 N2-amino group, whereas the other, 7B7-1, is less stringent and also recognizes several other modified metabolites, including bulky adducts at the N2-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 (4-MeIQx), 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (8-MeIQx), 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 3H-PhIP or 2-14C-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 3H-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 ∼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.

Original languageEnglish
Issue number11
Pages (from-to)2795-2806
Number of pages12
Publication statusPublished - 1995
Externally publishedYes

ID: 254776145