Determination of Aflatoxin B1–DNA Adduct in Rat Liver by Enzyme Immunoassay

Abstract

A simple, rapid and highly sensitive indirect competitive enzyme-linked 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, bovine serum albumin (BSA)–guanine– AFB ₁ . 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–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 produced 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 ≤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–AFB ₁ 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 AFB ₁ were used to validate the method. The AFB ₁ –DNA adduct formed in the liver tissue after 48 h of exposure was determined using the ELISA method developed. The liver AFB ₁ –DNA adduct ranged between 6.06 and 7.94 µg mg ^-1 DNA. The proposed method may find application in the biological monitoring of aflatoxin B ₁ in molecular epidemiological studies to assess the dietary exposure of aflatoxins.