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

Abstract

Methods for the synthesis of fumonisin B1 (FmB1) immunoconjugates and for the preparation of FmB1-tagged liposomes were developed and studied. Keyhole limpet hemocyanin-fumonisin B1 (KLH-FmB1) was synthesized using both the classical glutaraldehyde (GA) protocol and a modified 2-step GA protocol. The immunoconjugate was then used to raise polyclonal antibodies in Rambouilet sheep. The covalent coupling of FmB1 to the outer surface of the liposomes involved the derivatization of the FmB1 with a maleimide group which was allowed to react with a sulfhydryl group on the liposome surface. The maleimide group was introduced into the FmB1 via a hetero-bifunctional reagent sulfosuccinimidyl 4-[ N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC). The sulfhydryl group was generated by the deacetylation of an acetylthioacetate group that was incorporated into the lipid bilayer after the interaction of dipalmitoyl phosphatidyl ethanolamine (DPPE) with N-succinimidyl- S-acetylthioacetate (SATA). The sheep anti-FmB1 polyclonal antibodies as well as sulforhodamine B (SRB) dye-encapsulating, FmB1-tagged liposomes were used subsequently in the development of a Flow-Injection Liposome ImmunoAnalysis (FILIA) system for the detection of FmB1 in food.