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IndiaA specific and sensitive method to quantitate aflatoxin B1-N7-guanine adduct in urine samples by immunoaffinity chromatography coupled with indirect competitive enzyme-linked immunosorbent assay (ELISA) is reported. A novel in vitro method to synthesize an antigen (bovine serum albumin-guanine-aflatoxin B1) and its use to produce polyclonal antibodies specific to the hapten aflatoxin B-N7-guanine are discussed. An indirect competitive ELISA developed to quantitate aflatoxin adduct showed a 50% inhibition at 15.6 pmol aflatoxin B1-N7-guanine (y=66.73+ (-19.8)x, r=-0.997). Interference by aflatoxin B1 was less than 5%, and no interference by guanine, aflatoxin Gι and aflatoxin M1 was observed. An immunoaffinity column was also developed, by using these polyclonal antibodies, for single-step purification of aflatoxin B1-N7-gua-nine. The immunoaffinity column and the indirect competitive ELISA were evaluated and validated by quantitation of aflatoxin B1-N7-guanine in urine from rats dosed with aflatoxin B1 (1 mg/kg body weight). Spiking studies with standard aflatoxin B1-N7-guanine adduct at 2 and 4 μg/mL phosphate- buffered saline gave 96 and 100% recoveries, respectively, for immunoaffinity cleanup column. The method also was tested successfully for quantitating aflatoxin B1-N7-guanine adduct in spiked human urine. Recoveries of the adduct were 79-90%.

Quantitation of Aflatoxin B1-N7-Guanine Adduct in Urine by Enzyme-Linked Immunosorbent Assay Coupled with Immunoaffinity Chromatography