Aflatoxin B1 dihydrodiol antibody: production and specificity

A specific antibody for 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol) was prepared, and its reactivity was characterized for the major aflatoxin (AF) B1 (AFB1) metabolites. Reductive alkylation was used to conjugate AFB1-diol to ethylenediamine-modified bovine serum albumin (EDA-BSA) and horseradish peroxidase for use as an immunogen and an enzyme-linked immunosorbent assay (ELISA) marker, respectively. High reactant ratios, 1:5 and 1:10, for AFB1-diol-EDA-BSA (wt/wt) resulted in precipitated conjugates which were poorly immunogenic. However, a soluble conjugate obtained by using a 1:25 ratio of AFB1-diol to EDA-BSA could be used for obtaining high-titer AFB1-diol rabbit antibody within 10 weeks. Competitive ELISAs revealed that the AFB1-diol antibody detected as little as 1 pmol of AFB1-diol per assay. Cross-reactivity of AFB1-diol antibody in the competitive ELISA with AF analogs was as follows: AFB1-diol, 100%; AFB1, 200%; AFM1, 130%; AFB2a, 100%; AFG1, 6%; AFG2, 4%; aflatoxicol, 20%; AFQ1, 2%; AFB1-modified DNA, 32%; and 2,3-dihydro-2-(N7-guanyl)-3-hydroxy AFB1, 0.6%. These data indicated that the cyclopentanone and methoxy moieties of the AF molecule were the primary epitopes for the AFB1-diol antibody. The AFB1-diol competitive ELISA was subject to substantial interference by human, rat, and mouse serum albumins but not by BSA, Tris, human immunoglobulin G, or lysozyme. By using a noncompetitive, indirect ELISA with an AFB1-modified DNA solid phase, a modification level of one AFB1 residue for 200,000 nucleotides could be determined.