Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.