Development of an improved monoclonal antibody‐based Elisa for fumonisin b1–3and the use of molecular modeling to explain observed detection limits

Abstract

Monoclonal antibodies were prepared against the fumonisins, a group of mycotoxins produced by the plant pathogen, Fusarium moniliforme. Splenic lymphocytes, from Balb/c mice immunized with fumonisin B₁‐ovalbumin conjugate, were fused with SP2/O myeloma cells, and 14 hybridomas were selected. In a competitive enzyme‐linked immunosorbent assay, fumonisin B₁‐bovine serum albumin and free fumonisin B₁ (FB₁) competed for the monoclonal antibody. The concentrations of FB₁ required to inhibit 50% antibody binding (IC₅₀) ranged from 300 to 670 ppb. Antibodies also cross‐reacted with fumonisins B₂ and B₃ (FB₂, FB₃), and the hydrolyzed backbone of fumonisin B₁ (HB‐FB₁). None of the 14 monoclonal antibodies recognized the sphingolipids, sphingosine and sphinganine, that are structurally similar to the backbone of the fumonisins. Three‐dimensional computer models of FB₁, FB₂ and FB₃ show the amine backbone folding with the two esterified trimethyl‐propane‐1,2,3‐tricarboxylic acid side‐chains to form a cage into which the hydroxyl and acid groups of these fumonisins extend. The HB‐FB₁ molecule, with the two trimethyl‐propane‐1,2,3‐tricarboxylic acid esterified moieties at carbons 14 and 15 removed, does not possess two of the three branches which are folded together with inter‐hydrogen bonding to formulate the three‐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 cage of FB₁, attachment of the protein to the amine group, which is close to, or appears to be pari of, the epitope, may have allowed the immune system of the mouse to produce antibodies more specific for the FB₁‐protein conjugate than to free FB₁.