Quantitation of aflatoxin B1-N7-guanine adduct in urine by enzyme-linked immunosorbent assay coupled with immunoaffinity chromatography.
A 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 B1-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)chi; r = -0.997). Interference by aflatoxin B1 was less than 5%, and no interference by guanine, aflatoxin G1 and aflatoxin M1 was observed. An immunoaffinity column was also developed, by using these polyclonal antibodies, for single-step purification of aflatoxin B1-N7-guanine. 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 micrograms/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%.
Vidyasagar
T
T
University College of Science, Department of Biochemistry, Osmania University, Hyderabad, India.
Vyjayanthi
V
V
Sujatha
N
N
Rao
B S
BS
Bhat
R V
RV
D016604:Aflatoxin B1 / Q000031:analogs & derivatives* / Q000276:immunology / Q000652:urine; D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D003429:Cross Reactions; D018736:DNA Adducts; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006147:Guanine / Q000031:analogs & derivatives* / Q000652:urine; D006801:Humans; D007106:Immune Sera; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology; D017186:Titrimetry
D018736:DNA Adducts; D007106:Immune Sera; D012710:Serum Albumin, Bovine; D006147:Guanine; C028319:2,3-dihydro-2-(N(7)-guanyl)-3-hydroxyaflatoxin B1; D016604:Aflatoxin B1