The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Influence of the membrane on T-2 toxin toxicity in Saccharomyces spp.

In growing cells of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, T-2 toxin inhibits cell growth. We have examined the role of the yeast membranes in the uptake mechanism(s) of T-2 toxin. The effects of membrane-modulating agents, ethanol, cetyltrimethylammonium bromide, Triton X-100, and heat were studied; these agents were found to increase the sensitivity of the yeasts toward T-2 toxin. In the presence of 5% (vol/vol) ethanol, 2 micrograms of T-2 toxin per ml caused complete inhibition of growth. In the presence of 1 microgram of cetyltrimethylammonium bromide per ml, yeast cells became sensitive to T-2 toxin, starting with a concentration of 0.5 micrograms/ml. Triton X-100 at concentrations below 1% (vol/vol) sensitized the cells toward T-2 toxin, but at higher concentrations it protected the cells from T-2 toxin. Temperatures of incubation between 7 and 30 degrees C influenced the growth reduction caused by T-2 toxin. The greatest observed reduction of growth in T-2 toxin-treated cultures occurred at 30 degrees C. To further prove that the membrane influences the interaction of T-2 toxin with yeasts, we have studied a yeast mutant with a reduced plasma membrane permeability (G. H. Rank et al., Mol. Gen. Genet. 152:13-18, 1977). This yeast mutant proved to be resistant to T-2 toxin concentrations of up to 50 micrograms/ml. These results show that the membrane plays a significant role in the interaction of T-2 toxin with yeast cells.     

Metabolism of trichothecene mycotoxins. I. Microsomal deacetylation of T-2 toxin in animal tissues

In an attempt to elucidate the active form of T-2 toxin, one of trichothecene mycotoxins in vivo, the metabolism in animal tissues was studied in vitro by using gas liquid chromatography. T-2 toxin was selectively hydrolysed by the microsomal esterase at C-4, giving rise to HT-2 toxin as the only metabolite. This esterase activity was found mainly in the microsomes of liver, kidney, and spleen of laboratory animals. Since the enzymatic hydrolysis of T-2 toxin was inhibited by eserine, and diisopropylfluorophosphate, it is concluded that non-specific carboxyesterase [EC 3.1.1.1] of microsomal origin participates in this type of selective hydrolysis of T-2 toxin. The microsomal fraction from rabbit liver was proved to be a convinient material for the preparation of HT-2 toxin from T-2 toxin. From the evidence that the toxicity of HT-2 toxin is comparable to that of T-2 toxin and that the microsomal fraction of whole liver possesses the ability to biotransform the total lethal dose of T-2 toxin into HT-2 within a few minutes, T-2 toxin administered to animals is presumed to exhibit its toxicity partly as HT-2 toxin.     

Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-T-2 and <Emphasis Type="Italic">d</Emphasis><Subscript>3</Subscript>-HT-2 toxin

The type A trichothecenes T-2 and HT-2 toxins are toxic secondary metabolites produced by fungi of the Fusarium genus. Their occurrence in cereals, especially in oats, implies health risks for the consumer. Therefore, it is an important task to develop selective and sensitive methods for the analysis of T-2 and HT-2 toxins, and to undertake further studies on their stability and toxicity. Although most toxins are commercially available, their high prices are the limiting factor on the realization of these experiments. Thus, we developed a method for large-scale production of T-2 and HT-2 toxin as well as T-2 triol and T-2 tetraol. T-2 toxin was obtained in gram quantities by biosynthetic production with cultures of F. sporotrichioides. As HT-2 toxin was only formed as a by-product, and T-2 triol and T-2 tetraol were not generated, these compounds were produced by alkaline hydrolysis of T-2 toxin. Separation and isolation of crude toxins was achieved by fast centrifugal partition chromatography (FCPC), which is an efficient tool for the large-scale purification of natural products. Using this fast and yield effective technique, several hundred milligrams of HT-2 toxin, T-2 triol, and T-2 tetraol were obtained. Subsequent, HT-2 toxin and T-2 triol were used for the large-scale synthesis of isotope-labeled T-2 and HT-2 toxin, respectively. Using these standards, an isotope dilution-(ID)-HPLC-MS/MS method for the quantification of T-2 and HT-2 toxin in different matrices was developed.     

T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway

T-2 toxin, a member of the trichothecene mycotoxin family produced by the Fusarium fungi, has been shown to exert a variety of toxic effects on multiple targets in vivo. However, the embryonic toxicity of T-2 toxin in vitro remains unclear. In the present study, two permanent cell lines, embryonic stem cells (ES cells D3) and fibroblast 3T3 cells, were used to evaluate T-2 toxin toxicity. Differentiated mouse ES cells were cultivated as embryoid bodies along with T-2 toxin at different concentrations (0.5, 1, and 2 ng/ml) for 24 h. The increases in cellular reactive oxygen species (ROS), lipid and DNA oxidative damage, and loss of mitochondrial transmembrane potential were observed at 1 and 2 ng/ml concentrations. Flow cytometry showed that T-2 toxin induced cell cycle arrest and apoptosis. Furthermore, T-2 toxin opened the mitochondrial permeability transition pore, caused the release of cytochrome c from mitochondria and induced the upregulation of p53, caspase-9, caspase-3 expression and increased the ratio of Bax/Bcl-2. However, T-2 toxin-induced oxidative damage and apoptosis in differentiated ES cells decreased significantly in the presence of the antioxidant Trolox. Taken together, these results demonstrate that T-2 toxin induces oxidative stress and apoptosis in differentiated murine ES cells, and ROS-mediated mitochondrial pathway plays an important role in T-2 toxin induced apoptosis.     

A yeast bioassay for trichothecenes

Like all eucaryotic cells, yeasts are sensitive to trichothecenes, especially T-2 toxin and verrucarin A. Based on this sensitivity, a yeast bioassay was developed to evaluate the toxicity of corn samples. The bioassay was optimized using spiked maize extracts. The toxicity of samples was defined as toxicity equivalent to a certain concentration of T-2 toxin standards. The assay can be performed on crude extracts, but the results are more precise after column clean-up. The test can also be used for the screening of trichothecene toxicity in general. The relative standard deviation (RSD) at 85 % growth inhibition (EC85) was 4.5% for the T-2 toxin standards (n = 8). This corresponds to an initial T-2 toxin concentration of approximately 58 ppb in the corn sample. Samples containing 188 and 113 ppb T-2 toxin caused a growth inhibition higher than 85%, whereas samples with toxin concentrations of 56 and 19 ppb had a growth inhibition less than 85%. Therefore the test can be used for the qualitative evaluation of corn samples up to a level of 58 ppb +/- 2.8 ppb. The bioassay is easy to perform with minimum requirements for equipment. Results can be obtained within 24 h and a large number of samples can be analysed daily. The costs are low and the results obtained are repeatable. With some modifications this test can be used for toxicity studies on trichothecene metabolites as well as for extracts with unknown compounds with properties similar to trichothecenes.     

Neural Disturbances in Chickens Caused by Dietary T-2 Toxin

Graded concentrations of dietary T-2 toxin (0, 1, 2, 4, 8, and 16 mug/g) were fed to groups of 40 chickens. T-2 toxin was found to cause an abnormal positioning of the wings, hysteroid seizures, and impaired righting reflex in young chickens. The abnormal wing positioning occurred spontaneously or as the result of dropping from a height of 1 meter. The seizures could be elicited by rough handling or loud noises. The seizures and the abnormal wing posture would not occur again when the stimulus was repeated unless a rest period of 3 to 6 h was allowed. The loss of righting reflex could be demonstrated at any time. The total incidence of neural symptoms was dependent on the length of exposure to T-2 toxin and to its concentration. Neural toxicity occurred at dosages of 4, 8, and 16 mug per g of diet, which are the same doses that retard growth. This neural toxicity of T-2 toxin in chickens is similar to the neural disturbances associated with alimentary toxic aleukia, a nutritional toxicosis of humans produced by eating moldy grain. T-2 toxin has been implicated also in moldy corn toxicosis which has neural manifestations in horses and swine.     

Effect of mycotoxin T-2 on bioluminescence intensity of bacteria

The acute and chronic toxicity of T-2 was studied by bioluminescent method with the use of two strains of luminous bacteria--P. phosphorum Sq3 u V. fischeri F1 as biological objects. It was shown that in acute experiments after 10 min incubation of bacteria in the presence of T-2 the bioluminescence inhibition on the 50% level was observed at the toxin concentration equal to 12 mg/mL. In chronic experiments such a level of bioluminescence inhibition was registered after 16 hours incubation at the toxin concentration of 18 mg/mL. T-2 toxicity was also investigated in the presence of different serum albumin concentrations. It decreases with the increase of albumin concentration at the short term of incubation (5 min) of the mixture to be analyzed. In case of the longer term of incubation (up to 30 min) of this mixture T-2 toxicity was restored. Probably, it is a result of destruction of protein-toxin complex, which is, evidently, reversible and may be characterized by some index. It is necessary to emphasize that the sensitivity of T-2 analysis increases under the decrease of pH value up to lower bacterial physiological level, i.e. to 5-5.5. The revealed abilities of T-2 toxin effect on the intensity of bacterial bioluminescence may be used under the development of instrumental analytical approach on the basis of biosensor technology for testing this toxin in the environment. Taking into account the analysis simplicity and rapidity, such analytical device may have a perspective for wide practical application.     

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.     

Inhibition of mitogen-induced blastogenesis in human lymphocytes by T-2 toxin and its metabolites.

Concentrations of T-2, HT-2, 3'-OH T-2, 3'-OH HT-2, T-2 triol, and T-2 tetraol toxins which inhibited [3H]thymidine uptake in mitogen-stimulated human peripheral lymphocytes by 50% were 1.5, 3.5, 4.0, 50, 150, and 150 ng/ml, respectively. The results suggested that the initial hydrolysis of T-2 toxin and the hydroxylation of T-2 toxin to 3'-OH T-2 toxin did not significantly decrease the immunotoxicity of the parent molecule, whereas further hydrolysis to T-2 triol and T-2 tetraol toxins or hydroxylation to 3'-OH HT-2 toxin decreased in vitro toxicity for human lymphocytes.     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth.

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.     

Glucosylation and Other Biotransformations of T-2 Toxin by Yeasts of the Trichomonascus Clade

Trichothecenes are sesquiterpenoid toxins produced by Fusarium species. Since these mycotoxins are very stable, there is interest in microbial transformations that can remove toxins from contaminated grain or cereal products. Twenty-three yeast species assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including four Trichomonascus species and 19 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert the trichothecene T-2 toxin to less-toxic products. These species gave three types of biotransformations: acetylation to 3-acetyl T-2 toxin, glycosylation to T-2 toxin 3-glucoside, and removal of the isovaleryl group to form neosolaniol. Some species gave more than one type of biotransformation. Three Blastobotrys species converted T-2 toxin into T-2 toxin 3-glucoside, a compound that has been identified as a masked mycotoxin in Fusarium-infected grain. This is the first report of a microbial whole-cell method for producing trichothecene glycosides, and the potential large-scale availability of T-2 toxin 3-glucoside will facilitate toxicity testing and development of methods for detection of this compound in agricultural and other products.     

Lack of the Consensus Sequence Necessary for Tryptophan Prenylation in the ComX Pheromone Precursor

Recently we found that a single administration of T-2 toxin (T-2), a trichothecene mycotoxin, into mice induced DNA fragmentation, a biochemical hallmark of apoptosis, in the thymus.¹⁾ In this study, we investigated the effective chemical structure(s) of T-2-derived metabolites capable of inducing thymic apoptosis in vivo in mice. Metabolic conversion of T-2 to 3′-hydroxy-T-2 toxin (3′-OH-T-2) (Fig. 1) did not diminish the apoptosis-inducing activity, since essentially the same level of fragmented DNA was detected in the thymus taken from mice injected with either T-2 or 3′-OH-T-2. In contrast, hydrolysis of T-2 and 3′-OH-T-2 at the carbon-4 (C-4) position to HT-2 toxin (HT-2) and 3′-hydroxy-HT-2 toxin (3′-OH-HT-2), respectively, greatly decreased the level of DNA fragmentation. Similarly, hydrolysis of T-2 at the carbon-8 (C-8) position to neosolaniol strongly diminished its ability to induce DNA fragmentation. T-2 tetraol, having no ester groups, was unable to induce apoptosis. Based on the data presented in this study, we concluded that both the acetyl group at the C-4 position and the isovaleryl or 3′-hydroxyisovaleryl group at the C-8 position of the T-2 molecule are important for inducing cell death through apoptosis in the thymus.     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes.

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.