DEVELOPMENT OF AN IMPROVED MONOCLONAL ANTIBODY-BASED ELISA FOR FUMONISIN B-1-3 AND THE USE OF MOLECULAR MODELING TO EXPLAIN OBSERVED DETECTION LIMITS

Monoclonal antibodies were prepared against the fumonisins, a group of mycotoxins produced by the plant pathogen, Fusarium moniliforme. Sple nic lymphocytes, from Balb/c mice immunized with fumonisin B-1-ovalbum in conjugate, were fused with SP2/O myeloma cells, and 14 hybridomas w ere selected. In a competitive enzyme-linked immunosorbent assay, fumo nisin B-1-bovine serum albumin and free fumonisin B-1 (FB1) competed f or the monoclonal antibody. The concentrations of FB1 required to inhi bit 50% antibody binding (IC50) ranged fr-om 300 to 670 ppb. Antibodie s also cross-reacted with fumonisins B-2 and B-3 (FB2, FB3), and the h ydrolyzed backbone of fumonisin B-1 (HB-FB1). None of the 14 monoclona l antibodies recognized the sphingolipids, sphingosine and sphinganine , that are structurally similar to the backbone of the fumonisins. Thr ee-dimensional computer models of FB1, FB2 and FB3 show the amine back bone folding with the two esterified trimethyl-propane-1,2,3-tricarbox ylic acid sine-chains to form a cage into which the hydroxyl and acid groups of these fumonisins extend. The HB-FB1 molecule, with the two t rimethyl-propane-1,2,3-tricarboxylic acid esterified moieties at carbo ns 14 and 15 removed, does not possess two of the three branches which are folded together with inter-hydrogen bonding to formulate the thre e-dimensional structure that makes up the cage feature of FB1, FB2 and FB3. Because of the unexpected folding of the three arms to make a ca ge of FB1, attachment of the protein to the amine group, which is clos e to, or appears to be part of the epitope, may have allowed the immun e system of the mouse to produce antibodies more specific for the FB1- protein conjugate than to free FB1.