Monoclonal antibody to aflatoxin B1-modified DNA detected by enzyme immunoassay

Abstract

Monoclonal antibodies were obtained after fusion of mouse P3 X 63 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin B1-adducted DNA complexed with methylated bovine serum albumin. Selected hybridomas were found to produce monoclonal antibodies specific for aflatoxin B1-modified DNA containing both the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'oxo-N5-pyrimidyl) -3-hydroxyaflatoxin B1, suggesting that these DNA adducts share a common antigenic determinant. The monoclonal antibody was not reactive towards the free aflatoxin B1-guanine adducts in solution, seven other aflatoxin derivatives, or benzo[a]pyrene-adducted DNA. A noncompetitive ultrasensitive enzyme radioimmunoassay could measure 15 fmol of aflatoxin B1-DNA adducts in 10 ng of DNA and was at least 100-fold more sensitive than the standard enzyme-linked immunosorbent assay. Competitive enzyme-linked immunosorbent assay with these monoclonal antibodies reliably quantitated aflatoxin B1 adducted in vivo to rat liver DNA at adduct levels of one aflatoxin B1 residue per 250,000 nucleotides. The competitive ultrasensitive enzyme radioimmunoassay was determined to be at least 6-fold more sensitive than the competitive enzyme-linked immunosorbent assay in analysis of aflatoxin B1-adducted DNA. Therefore, enzyme immunoassay using monoclonal antibodies will be useful analytical tools for studying both the molecular interactions of aflatoxin B1 with DNA and the occurrence of aflatoxin B1-DNA adducts in biological specimens from people exposed to this environmental carcinogen.