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 IgG₁ and IgG₃ antibodies were developed tothe food mutagens, 2-amino-1-methyl-6-phenylimidazo4,5-b]pyridine(PhIP) and 2-amino-3,4,8-trimethylimidazo4,5-f]quinoxaline(4,8-DiMeIQx) in order to make specific and sensitive detectionand purification systems suitable for biological samples. Theantibodies were developed with the strategy that cross-reactionwith analogues modified in the N²-position was desirable. Competitiveenzyme-linked immunosorbent assays (ELISA) with 50% inhibitionby 0.4–6 pmol food mutagen were developed. The epitopesrecognized by the antibodies have been characterized by ELISAusing 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx,and other food mutagens. One of the anti-PhIP antibodies onlyrecognizes PhIP and those PhIP-analogues which have minor modificationsin the N²-amino group, whereas the other, 7B7-1, is less stringentand also recognizes several other modified metabolites, includingbulky adducts at the N²-amino group e.g. the major guanine anddeoxyguanosine adducts isolated from PhIP-modified DNA. Theantibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo4,5-f]quinoxaline(4-MeIQx), 2-amino-3, 8-dimethylimidazo4, 5-f]quinoxaline (8-MeIQx),and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two ofthese antibodies only bind analogues with minor modificationsin the free amino group, whereas analogues with major modificationsin this position, including a deoxyguanosine adduct, react withthe third antibody. Urine samples and faecal extracts from ³H-PhIPor 2-¹⁴C-DiMeIQx dosed rats were analysed by these ELISA assays,and high correlations between radioactivity and response inthe ELISA assays were observed. Urine samples and faecal extractsfrom ³H-PhIP-dosed rats were purified on an affinity columncontaining the less stringent anti-PhIP antibody, 7B7-1. Theaffinity column was found by high performance liquid chromatography(HPLC) analysis to concentrate exclusively labelled material.This affinity column also bound PhIP-related materials fromdilute samples of acid hydrolysed PhIP-DNA with high efficiency.Only