Enzyme-Linked Immunosorbent Assay for T-2 Toxin Metabolites in Urine

Abstract

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.

Simultaneous occurrence of fumonisin B1 and other mycotoxins in moldy corn collected from the People's Republic of China in regions with high incidences of esophageal cancer

“…For the determination of total type-A TCTC, an indirect ELISA was used. This assay quantitatively determines all of the type-A TCTC after converting them to T-2 tetraacetate (15,24). The analytical recoveries of T-2 toxin added to a blank corn sample at 0.1-, 0.5-, 2-, and 5-ppm levels were 123, 94, 98, and 83.5%, respectively.…”

“…The Sep-Pak C-18 reverse-phase cartridge was obtained from Waters Associates (Milford, Mass.). Specific antibodies against mycotoxins and related immunochemical reagents were prepared in our laboratory (4,15,31). All other chemicals and organic solvents were either analytical grade or high-performance liquid chromatography (HPLC) grade.…”

“…The toxins were extracted from the samples with 50% MeCN in a manner similar to that for FB, analysis. For total type-A TCTC, an enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies that have a wide specificity to most type-A TCTC was used (15,24). The samples were subjected to hydrolysis and acetylation before ELISA.…”

In vitrosynthesis of a type A trichothecenes complete antigen from T-2 toxin

“…Methods that can simultaneously detect all type A trichothecenes are rare. Wei and Chu (1987), Fan et al (1988) and Lee, Wei, and Chu (1989) reported immunochemical methods to detect type A trichothecenes, but none of these methods can detect all type A trichothecenes. Our research has extended these findings by further optimizing the preparation of an antigen to type A trichothecene skeletons.…”

Natural occurrence of Fusarium mycotoxins in field samples from the 1992 Wisconsin corn crop

“…Fusarium tricinctum 696 (now reclassified as F. sporotrichioides) in cracked corn at 15ЊC under the conditions described earlier (10). Acetyl-T-2 toxin (Ac-T-2), T-2 tetraol tetraacetate (T-2-4ol-4Ac), 3-Ac-NEOS, hemisuccinate (HS) of 3-Ac-NEOS, and 3-Ac-NEOS-HS-horseradish peroxide (HRP) conjugate were prepared according to methods described previously (20,41). DON was produced by F. graminearum NRRL 5883 in cracked corn at 25ЊC under the conditions described by Vesonder et al (39).…”