T. Vidyasagar et al., QUANTITATION OF AFLATOXIN B-1-N-7-GUANINE ADDUCT IN URINE BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY COUPLED WITH IMMUNOAFFINITY CHROMATOGRAPHY, Journal of AOAC International, 80(5), 1997, pp. 1013-1022
A specific and sensitive method to quantitate aflatoxin B-1-N-7-guanin
e adduct in urine samples by immunoaffinity chromatography coupled wit
h indirect competitive enzyme-linked immunosorbent assay (ELISA) is re
ported, A novel in vitro method to synthesize an antigen (bovine serum
albumin-guanine-aflatoxin B-1) and its use to produce polyclonal anti
bodies specific to the hapten aflatoxin B-1-N-7-guanine are discussed,
An indirect competitive ELISA developed to quantitate aflatoxin adduc
t showed a 50% inhibition at 15.6 pmol aflatoxin B-1-N-7-guanine (y =
66.73 + (-19,8)x; r = -0.997). Interference by aflatoxin B-1 was less
than 5%, and no interference by guanine, aflatoxin G(1) and aflatoxin
M-1 was observed, An immunoaffinity column was also developed, by usin
g these polyclonal antibodies, for single-step purification of aflatox
in B-1-N-7-guanine, The immunoaffinity column and the indirect competi
tive ELISA were evaluated and validated by quantitation of aflatoxin B
-1-N-7-guanine in urine from rats dosed with aflatoxin B-1 (1 mg/kg bo
dy weight), Spiking studies with standard aflatoxin B-1-N-7-guanine ad
duct at 2 and 4 mu g/mL phosphate-buffered saline gave 96 and 100% rec
overies, respectively, for immunoaffinity cleanup column, The method a
lso was tested successfully for quantitating aflatoxin B-1-N-7-guanine
adduct in spiked human urine. Recoveries of the adduct were 79-90%.