Determination of aflatoxin B1-DNA adduct in rat liver by enzyme immunoassay.
A simple, rapid and highly sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (AFB1)-DNA adducts is reported. Polyclonal antibodies specific to the aflatoxin B1-N7-guanine adduct were produced using a novel synthetic antigen, bovine serum albumin (BSA)-guanine-AFB1. The antibodies were characterized by the Ouchterlony double diffusion technique and by antibody capture assay. The working range of the indirect competitive assay developed was between 0.45 and 330 ng of the analyte [calf thymus (CT)-DNA-AFB1]. A 50% inhibition was attained at 15 ng of the analyte (CT-DNA-AFB1). The antibody capture assay indicated that the antibody produced cross-reacted 100, 92 and 110% with BSA-guanine-AFB1, CT-DNA-AFB1 and CT-DNA-formamidopyrimidine-AFB1, respectively. When free AFB1 and guanine were used as competing analytes, the antibodies showed < or = 5% and zero cross-reactivity at the 50% inhibition level. Spiking studies indicated a recovery in the range 96-97 and 74-78% when standard CT-DNA-AFB1 was added to 10 mM phosphate buffer (pH 7.2) and control rat liver tissue, respectively. Rats exposed to a single oral dose of 1 mg kg-1 body mass of pure AFB1 were used to validate the method. The AFB1-DNA adduct formed in the liver tissue after 48 h of exposure was determined using the ELISA method developed. The liver AFB1-DNA adduct ranged between 6.06 and 7.94 micrograms mg-1 DNA. The proposed method may find application in the biological monitoring of aflatoxin B1 in molecular epidemiological studies to assess the dietary exposure of aflatoxins.
Vidyasagar
T
T
Department of Biochemistry, University College of Science, Osmania University, Hyderabad, India.
Sujatha
N
N
Sashidhar
R B
RB
D016604:Aflatoxin B1 / Q000032:analysis*; D000818:Animals; D000941:Antigens / Q000276:immunology; D002273:Carcinogens / Q000032:analysis*; D002417:Cattle; D018736:DNA Adducts; D004797:Enzyme-Linked Immunosorbent Assay; D006147:Guanine; D008099:Liver / Q000737:chemistry*; D008297:Male; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D012710:Serum Albumin, Bovine
D000941:Antigens; D002273:Carcinogens; D018736:DNA Adducts; D012710:Serum Albumin, Bovine; D006147:Guanine; D016604:Aflatoxin B1