Preparation of reagents for the determination of fumonisin B1 by flow-injection immunoanalysis

Methods for the synthesis of fumonisin B1 (FmB1) immunoconjugates and for t he preparation of FmB1-tagged liposomes were developed and studied. Keyhole limpet hemocyanin-fumonisin B1 (KLH-FmB1) was synthesized using both the c lassical glutaraldehyde (GA) protocol and a modified 2-step GA protocol. Th e immunoconjugate was then used to raise polyclonal antibodies in Rambouile t sheep. The covalent coupling of FmB1 to the outer surface of the liposome s involved the derivatization of the FmB1 with a maleimide group which was allowed to react with a sulfhydryl group on the liposome surface. The malei mide group was introduced into the FmB1 via a hetero-bifunctional reagent s ulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMC C). The sulfhydryl group was generated by the deacetylation of an acetylthi oacetate group that was incorporated into the lipid bilayer after the inter action of dipalmitoyl phosphatidyl ethanolamine (DPPE) with N-succinimidyl- S-acetylthioacetate (SATA). The sheep anti-FmB1 polyclonal antibodies as we ll as sulforhodamine B (SRB) dye-encapsulating, FmB1-tagged liposomes were used subsequently in the development of a Flow-Injection Liposome ImmunoAna lysis (FILIA) system for the detection of FmB1 in food. (C) 2000 Elsevier S cience B.V. All rights reserved.