Monoclonal antibodies to 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) and their use in the analysis of well-done fried beef

Abstract

Four monoclonal antibodies, PhIP-1, -2, -3 and -4, have been produced that bind to PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), a mutagenic aromatic amine produced in meat by cooking. The immunogen was an analog of PhIP conjugated to a carrier protein through the 2-amino group of PhIP, leaving the pyridine and phenyl rings unmodified. The antibodies show a range of binding specificities for PhIP and its structural analogs, with PhIP-1 being the most highly selective, requiring the entire PhIP molecule with the exception of the exocyclic amino group to be present for recognition. A competition enzyme linked immunosorbent assay (ELISA) incorporating these antibodies was developed and applied to the analysis of fried meat extracts differing in mutagen content. In extracts of fried beef, more PhIP-like material was found than would be expected based on chemical estimates of the amount of PhIP, suggesting that frying produces other compounds that are structurally similar to PhIP. These PhIP-Like compounds were separated from PhIP using HPLC. Addition of creatinine to the beef prior to frying increased both the mutagenicity of the extracts and the amount of PhIP-Like material. Microwave pret-reatment prior to frying reduced both immunochemical reactivity and mutagenicity.