QUANTITATION OF AFLATOXIN B-1-N-7-GUANINE ADDUCT IN URINE BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY COUPLED WITH IMMUNOAFFINITY CHROMATOGRAPHY

Citation
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
Citations number
24
Categorie Soggetti
Chemistry Analytical
ISSN journal
10603271
Volume
80
Issue
5
Year of publication
1997
Pages
1013 - 1022
Database
ISI
SICI code
1060-3271(1997)80:5<1013:QOABAI>2.0.ZU;2-#
Abstract
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%.