Detection of the mycotoxin fumonisin B1by a combination of immunofluorescence and capillary electrophoresis

Abstract

The specificity of antibodies has been combined with the speed and resolving power of capillary electrophoresis for application to the analysis of the mycotoxin fumonisin B₁(FB₁). The assay was based upon the competition between unlabeled FB₁ (i.e. from a sample) and a fluorescein‐labeled FB₁ reagent (FB₁‐FL). The FB₁‐FL was prepared by derivatizing FB₁ with fluorescein isothiocyanate and was purified with affinity columns consisting of a monoclonal antibody (MAb) directed against fumonisins (clone P2A5–3‐F3) coupled to agarose. The purified FB₁‐FL was subjected to capillary zone electrophoresis. Addition of purified MAb to FB₁‐FL before separation resulted in the formation of a complex (MAb.FB₁‐FL) with resulting quenching of fluorescence and decrease in the intensity of the FB₁‐FL peak. When unlabeled FB₁ was also added to the reaction mixture the FB₁ and FB₁‐FL competed for the limited amount of antibody present causing the FB₁‐FL peak to increase in direct proportion to the amount of unlabeled FB₁ present. Fumonisin standards could be analyzed with this technique with a total analysis time of 6 min, 2 min of which was required for washing the capillary between analyses. The concentration of unlabeled FB₁ required to obtain 50% of the maximum fluorescence (IC₅₀) was highly dependent upon the antibody concentration and ranged from 58 to 4170 ng ml^‐1 at 15–75 μg m^‐1 of antibody. The optimum performance was seen with 25–50 μg ml^‐1 of antibody, with IC₅₀’s between 500 and 1700 ng ml^‐1 of FB₁. The technique was applied to a limited number of corn samples spiked with high levels of FB₁(> 10 ppm). This technology holds considerable promise for the rapid analysis of mycotoxins in foods.