DETERMINATION OF AFLATOXIN B-1-DNA ADDUCT IN RAT-LIVER BY ENZYME-IMMUNOASSAY

A simple, rapid and highly sensitive indirect competitive enzyme-linke d immunosorbent assay (ELISA) to determine aflatoxin B-1 (AFB(1))-DNA adducts is reported, Polyclonal antibodies specific to the aflatoxin B -1-N-7-guanine adduct were produced using a novel synthetic antigen, b ovine serum albumin (BSA)-guanine-AFB(1). The antibodies were characte rized by the Ouchterlony double diffusion technique and by antibody ca pture assay. The working range of the indirect competitive assay devel oped was between 0.45 and 330 ng of the analyte [calf thymus (CT)-DNA- AFB(1)]. A 50% inhibition was attained at 15 ng of the analyte (CT-DNA -AFB(1)). The antibody capture assay indicated that the antibody produ ced cross-reacted 100, 92 and 110% with BSA-guanine-AFB(1), CT-DNA-AFB (1) and CT-DNA-formamidopyrimidine-AFB(1), respectively. When free AFB (1) and guanine were used as competing analytes, the antibodies showed less than or equal to 5% and zero cross-reactivity at the 50% inhibit ion level, Spiking studies indicated a recovery in the range 96-97 and 74-78% when standard CT-DNA-AFB(1) was added to 10 mM phosphate buffe r (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 AFB(1) were used to validate the method, The AFB(1)-DNA adduct formed in the liver tiss ue after 48 h of exposure was determined using the ELISA method develo ped. The liver AFB(1)-DNA adduct ranged between 6.06 and 7.94 mu g mg( -1) DNA. The proposed method may find application in the biological mo nitoring of aflatoxin B-1 in molecular epidemiological studies to asse ss the dietary exposure of aflatoxins.