INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Deoxynivalenol (DON) (vomitoxin) (Fig. 1A) is one of the sesquiterpene mycotoxins classified as 12,13-epoxy-trichothecenes (28). This compound occurs naturally in infected corn (19, 29), small grains (18, 31), and mixed feeds (29). DON is mainly produced by the fungus Gibberella zeae (Schwein.) Petch (anamorph, Fusarium graminearum Schwabe). At the cellular level, the main toxic effect of DON is inhibition of protein synthesis via binding to ribosomes and interfering with peptidyltransferase (4, 42). In animals, DON can cause anorexia and emesis (vomiting) (37). Other toxic effects of DON include skin irritation, hemorrhaging, hematological changes, human lymphocyte blastogenesis impairment, radiomimetic effects, apoptosis (cytotoxicity), and immunotoxicity (37).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Structural comparison of DON and nivalenol with DON mimotope peptide SWGPFPF (DONPEP.2). (A) Two-dimensional representation of DON. The asterisk indicates the site of conjugation of the carrier protein (e.g., BSA) to DON. (B) Two-dimensional structure of nivalenol, an analog of DON whose structure is known. (C) Three-dimensional structural model of the DON mimotope peptide. The white spheres represent oxygen atoms, the white cylinders represent nitrogen atoms, and the grey cylinders represent carbon atoms in panels C through E. (D) Three-dimensional stereo view of the crystallographic structure of nivalenol (CSD entry DUTJOR10 [4a]). (E) Stereo view of the optimal PowerFit superposition of the known nivalenol structure and the DON mimotope peptide structure. Nivalenol aligns with the peptide model main-chain atoms between residues 2 and 5 (TrpGlyProPhe) and partially overlaps the side chains or Trp-2 and Pro-4.

A major way to eliminate DON from human and animal food is to detect contaminated raw materials and divert them from feed and finished food. Compared with other analytic methods, immunoassays have several advantages for rapid field testing, including high specificity, sensitivity, facile sample preparation, and ease of use (33). Following the development of the first monoclonal antibody (mAb) to DON (6), immunological methods, mainly an enzyme-linked immunosorbent assay (ELISA), have been used widely for detection of DON (33). Unfortunately, the efficiency of chemical conjugation of DON to a carrier protein or an enzyme is low because such conjugation involves extensive modification and blocking stages and causes substantial bridge group interference and unwanted cross-reactions (6, 33, 45). Also, when DON is conjugated to a carrier protein, it is weakly immunogenic. Finally, since DON is toxic and is included as a standard and conjugate in immunoassay mixtures, it may pose a toxicity risk to kit users.

One possible alternative to using mycotoxins as immunochemical reagents is to develop protein or peptide mimics that serve the same function. One approach for doing this is via generation of anti-idiotype antibodies (7, 9, 23), whose structures mimic the surface structures of low-molecular-weight biological toxins. Sometimes the sensitivity of the original antibody can be improved by generating anti-anti-idiotype antibodies (8). However, these techniques are time-consuming and costly. A new technique, phage display, allows foreign peptides and proteins to be genetically fused to the N terminus of minor coat protein g3p of the filamentous phage fd, which results in display of the peptides and proteins on the surface of the virion (40). Since random oligonucleotide sequences are inserted into the g3 gene of filamentous phage fd, phage display provides a way to construct extensive peptide libraries that may be screened in order to select peptides with specific affinities or activities (14, 15, 39). Phage-displayed short peptide libraries have been widely used in a number of applications (12, 24), including epitope mapping (14, 39), mapping of protein-protein contacts (22), identification of protease substrates (41), and identification of integrin (32) and other receptors (16), antagonists (35), and ligands (24). Only a few workers have used this technology to select peptide mimics of nonproteinaceous chemicals other than biotin (43) and carbohydrates (21).

Given the feasibility of selecting peptides that mimic nonproteinaceous chemicals, we were interested in determining whether peptide mimics of low-molecular-weight mycotoxins could be identified. Once selected, these peptide mimics might be useful in mycotoxin immunoassays and for developing new antibodies against mycotoxins. In this paper we describe two closely related heptapeptide mimotopes for DON that were selected from a phage-displayed random peptide library and the use of these mimotopes in an analysis of DON by ELISA. One of these mimotope peptides was synthesized and compared with DON for use in immunoassays, in cytotoxicity studies of cell cultures, and in in vitro protein synthesis inhibition studies.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents. All inorganic chemicals and organic solvents were reagent grade or better. Crude ovalbumin, bovine serum albumin (BSA) (fraction V), 3,3',5,5'-tetramethylbenzidine, phenylmethylsulfonyl fluoride, polyoxyethylene sorbitan monolaurate (Tween 20), DON, mouse immunoglobulin G (IgG), and goat anti-mouse IgG peroxidase conjugate were purchased from Sigma Chemical Co. (St. Louis, Mo.). Dimethyl sulfoxide and methanol were purchased from J. T. Baker Inc. (Phillipsburg, N.J.), and nonfat dry milk was obtained from Commander Foods, Inc., (Syracuse, N.Y.). DON-BSA, DON-ovalbumin, and DON-horseradish peroxidase (HRP) conjugates at DON's 3-hydroxyl group were prepared by using an activated N-hydroxysuccinimide ester as described by Casale et al. (6) and were kindly provided by Frank E. Klein (Neogen Corporation, Lansing, Mich.). Sheep anti-M13 HRP conjugate was purchased from Pharmacia Biotech Inc. (Piscataway, N.J.). m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS) and p-nitrophenyl phosphate disodium salt tablets were purchased from Pierce Chemical Company (Rockford, Ill.). Anti-DON mAb from hybridoma cell line 6F5 was prepared as described previously (6).

Phage selection by panning-elution. A phage-displayed heptapeptide library with 2 × 10⁹ sequence complexity (random peptide 7-mers fused to minor coat protein g3p of filamentous coliphage M13) was purchased from New England Biolabs, Inc. (Beverly, Mass.). After amplification in Escherichia coli ER2537 (New England Biolabs, Inc.), the phages in the library were selected by panning-elution as described below.

Competitive ELISA performed with phage-displayed peptide. To perform a competitive direct ELISA (CD-ELISA) with phage-displayed peptide, Immuno-4 microtiter wells were coated with mAB 6F5 and blocked as described above for panning-elution selection. Various concentrations of DON (0 to 10,000 ng/ml in 1% methanol-PBS) were mixed with equal volumes of phage-displayed peptide (diluted 1:10 in 10% milk-PBS). The mixtures were added to mAB 6F5-coated microtiter wells (100 µl/well), and the preparations were incubated at 37°C for 1 h. After the wells were washed six times with PBS-T (300 µl/well), the amounts of bound recombinant phage were determined by incubating the preparations with 100 µl of sheep anti-M13 HRP conjugate (diluted 1:5,000 in 10% milk-PBS) per well at 37°C for 1 h. Amounts of bound enzyme were determined as described above. For comparison, 50 µl of DON-HRP per well was also mixed with 50 µl of DON at various concentrations (0 to 10,000 ng/ml in 1% methanol-PBS) per well, and the preparations were incubated in mAB 6F5-coated microtiter wells at 37°C for 1 h. Amounts of bound enzyme were determined as described above.

DNA sequencing. After specific binding to mAB 6F5 was confirmed by ELISA, 10 ml of recombinant phage particles (10¹¹ PFU/ml in Luria-Bertani medium) from each positive phage clone was used for single-stranded DNA isolation performed with a QIAprep Spin M13 kit (QIAGEN Inc., Chatsworth, Calif.). The single-stranded DNA was sequenced with 28 gIII sequencing primer and 96 gIII sequencing primer (New England Biolabs, Inc.) by using Taq cycle sequencing and dye terminator chemistry at the Michigan State University DNA Sequencing Facility.

Translational fusion with bacterial AP. A 179-bp DNA fragment encoding the g3p signal peptide, DONPEP.2, and the first 17 amino acids of M13 phage g3p protein was amplified by PCR performed with Pfu DNA polymerase, a sense primer (5'-GCCAAGCTTAGATCTTGGAGCCTTTTTTTTGGAG-3'), and an antisense primer (5'-CCGGTCGACCTGTATGGGATTTTGCTAAACAACT-3'). After gel purification, the amplified DNA was digested with BglII and SalI and was cloned into BamHI-SalI-digested pLIP5 (5), which generated plasmid pQY7. The ligated product was used to transform E. coli DH11S competent cells (Gibco BRL, Gaithersburg, Md.), which generated DH11S/pQY7. DONPEP-alkaline phosphatase (AP) fusion protein was produced from DH11S/pQY7 in SB medium (35 g of Bacto Tryptone/liter, 20 g of Bacto Yeast Extract/liter, 5 g of NaCl/liter) containing ampicillin (100 µg/ml) and was induced by 1 mM isopropyl--D-thiogalactopyranoside (IPTG). The periplasmic DONPEP-AP fusion protein was extracted by suspending the bacterial cells (1:5, vol/vol) in lysis buffer (50 mM Tris-HCl [pH 8.0], 20% sucrose, 10 mM EDTA, 0.1 mg of lysozyme per ml, 0.5 mM phenylmethylsulfonyl fluoride); the preparation was left on ice for 1 h with agitation before centrifugation for 30 min at 7,000 × g. The supernatant was filtered through a 0.4-µm-pore-size porous filter. The activity of the DONPEP-AP fusion protein was determined by CD-ELISA.

CD-ELISA performed with peptide C430 and its HRP conjugate. A DON mimotope peptide with a structurally flexible linker and a cysteine residue (NH₂-SWGPFPFGGGSC-COOH) was synthesized via [N-(9-fluorenylmethoxycarbonyl)] Fmoc) chemistry at Bio-Synthesis, Inc. (Lewisville, Tex.); this compound was designated C430. Synthetic peptide C430, at various concentrations, was used to compete with DON-HRP for binding to mAB 6F5 in a CD-ELISA. C430 was also chemically conjugated to HRP with a sulfo-MBS cross-linker. The procedure used for the CD-ELISA performed with the C430-HRP conjugate was the same as the procedure used for the CD-ELISA performed with DON-HRP described above, except that C430-HRP (diluted 1:5,000 in blocking buffer) replaced DON-HRP.

Immunization of animals with C430-BSA conjugate. Synthetic peptide C430 was conjugated to BSA with a sulfo-MBS cross-linker. The conjugate (molar ratio of peptide C430 to the carrier protein BSA, approximately 29:1) was used to inject 7-week-old BALB/c female mice intraperitoneally or 6-month-old New Zealand White rabbits subcutaneously. The initial injections used for mice contained 100 to 200 µg of C430-BSA conjugate in 200 µl of saline-Freund's complete adjuvant (1:1); these injections were followed at 3-week intervals by boosters consisting of 100 to 200 µg of conjugate in 200 µl of saline-Freund's incomplete adjuvant (1:1). The initial inoculum used for rabbits consisted of 1 mg of C430-BSA conjugate in 1 ml of saline-Freund's complete adjuvant (1:1); this was followed at 4-week intervals by boosters consisting of 250 µg of conjugate in 1 ml of saline-Freund's incomplete adjuvant (1:1). Animals were bled 1 week after each booster injection. Antisera were screened for specific binding in phage-displayed DONPEP.2-coated wells by a competitive indirect ELISA in which C430 or DON was used as the competitive antigen.

Cytotoxicity and effects on protein synthesis in vitro. Ten-week-old B6C3F1 mice were euthanized by cervical dislocation, and the femurs were removed. Bone marrow cells were flushed from the femurs by using a 1-ml syringe and a 25-gauge needle. Erythrocytes were lysed with 0.83% ammonium chloride. Cell number and viability were determined by trypan blue dye exclusion by using a hemacytometer. The cells were cultured at a concentration of 10⁶ cells/ml in 96-well flat-bottom plates in RPMI-1640 in a humidified incubator containing 5% CO₂. RPMI-1640 was supplemented with 100 U of penicillin per ml, 100 µg of streptomycin per ml, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 2 mM glutamine, and 10% fetal bovine serum. DON and synthetic peptide C430 were diluted in RPMI-1640. Duplicate cultures were treated. After 18 h of exposure to DON and synthetic peptide C430, cell viability was determined by using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] conversion assay as described by Marin et al. (26).

Homologous sequence search and peptide modeling. The computer program Sequery (10, 13) was used to search for sequences similar to the DONPEP.2 peptide sequence (SWGPFPF) in a database of nonhomologous protein structures derived from the 25% identity set of the Protein Data Bank (PDB) Select list (20). Sequery identified all occurrences of protein sequences in the PDB that matched tetrapeptidyl fragments of DONPEP.2. The following conserved residue substitutions were allowed during the sequence search: S for T, W for F, P for ST, and F for ST. The resulting sequence analogs consisted of tetrapeptides that matched a portion of DONPEP.2 peptide and had known three-dimensional structures available in the Brookhaven PDB (1, 3). The secondary structures of these analogs were subsequently analyzed by using the Superpositional Structural Assignment computer program (13). This program superimposed the analogs onto a set of regular secondary structure templates and assigned each analog to the structural category (e.g., -helix, -strand, reverse turn) which it matched most closely within a 1.0-Å main-chain root-mean-square positional deviation (RMSD); if no template matched within a 1.0-Å RMSD, the structure of the analog was considered irregular. The analogs that were found to be irregular were analyzed visually by using molecular graphics, so that slightly irregular structures (e.g., a bent -helical turn) could be assigned to the most appropriate structural category.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Panning-elution selection of specific phages. After each round of panning-elution selection, the number of eluted phage was determined. Enrichment for specific phages with affinity to mAb 6F5 was observed after two rounds of panning-elution selection. The phages with affinity to mAb 6F5 were enriched approximately 6 orders of magnitude after the fourth round of panning-elution. Fifteen individual phage plaques from the fourth-round-selected phages were randomly isolated and used to infect E. coli ER2537 for phage reamplification. Each of the 15 phage isolates was tested to determine its binding to mAb 6F5 by ELISA. Five of these phage isolates, designated DONPEP.1, DONPEP.2, DONPEP.3, DONPEP.4, and DONPEP.12, exhibited binding to mAb 6F5.

Use of DON mimotope peptide in competitive ELISA. To determine whether the positive recombinant phage peptides actually mimicked the epitope recognized by mAB 6F5 or just bound nonspecifically to the surface of the antibody molecule outside the antigen binding site, two different positive phage clones were tested for binding to mAB 6F5 by performing a CD-ELISA (Fig. 2). Binding of phage clones DONPEP.2 and DONPEP.12 to immobilized mAB 6F5 was competitively inhibited by free DON. This strongly suggested that these two phage clones bound to the antigen binding site of the mAb, mimicking, in part, the structural epitope of DON.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Competition between phage-displayed mimotope peptides and DON for binding to immobilized mAB 6F5 in CD-ELISA. Various concentrations of free DON competed with equal volumes of phage-displayed peptides (at a constant concentration) for binding to immobilized mAB 6F5. Bound phage peptide was detected with HRP-conjugated sheep anti-M13 IgG and then measured by absorbance. DON-HRP was included as a positive control.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Competition between synthetic peptide C430 and DON for binding to mAB 6F5. (A) DON and synthetic peptide C430 competing with DON-HRP for binding to immobilized mAB 6F5. (B) DON and synthetic peptide C430 competing with C430-HRP for binding to immobilized anti-DON mAB 6F5.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   CD-ELISA performed with DONPEP-AP fusion protein. Binding of the DONPEP-AP fusion protein to immobilized mAB 6F5 was inhibited by free DON. Competition of free DON with DON-HRP was used as a control.

CD-ELISA for wheat extract spiked with DON. To test the feasibility of using DON mimotope peptide sequences as immunochemical reagents for DON immunoassays in food and feed, a CD-ELISA was performed with wheat extracts spiked with DON. Both C430-HRP conjugate and DONPEP-AP fusion protein exhibited binding to immobilized mAB 6F5 in the wheat extract (Fig. 5). All three conjugates produced similar linear inhibition curves at DON concentrations ranging from 0.1 to 10 µg/ml in wheat extract. However, a slightly lower level of absorbance was observed in the CD-ELISA performed with C430-HRP and DONPEP-AP in wheat extract than in PBS buffer, indicating that the wheat extract interfered with binding of the mimotope peptide to anti-DON mAB 6F5 to some degree.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Use of C430-HRP and DONPEP-AP in a DON immunoassay (CD-ELISA) performed with wheat extract spiked with DON. Immulon-4 microtiter wells were coated with mAB 6F5 antibody, and DON-HRP was used as a positive control.

Immunization with C430-BSA conjugate. If the selected peptides represent a true image of the DON surface structure, they might elicit an immunoresponse similar to that of the original DON epitope and, therefore, serve as alternative antigens for DON. When rabbits and mice were immunized with C430-BSA conjugate, both types of animals produced antibody specific to the DON mimotope peptide sequence after the second injection of C430-BSA. After four injections, the antiserum exhibited a strong antibody response against the DON mimotope peptide sequence (Fig. 6). A synthetic peptide C430 concentration of 0.39 µM resulted in 50% inhibition of antiserum binding to the immobilized phage-displayed peptide. However, binding of antisera to immobilized phage-displayed peptide was not inhibited by free DON in solution, indicating that antibodies in the immunized animals were not specific for DON.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Specificity of antibody from C430-BSA-immunized mice. DON and C430 at various concentrations were used to inhibit the binding of antisera to immobilized DONPEP.2 phage. Bound mouse antibodies were detected with HRP-conjugated goat anti-mouse IgG, and the amounts of these antibodies were measured by absorbance.

Cytotoxicity and effects on protein synthesis in vitro. One of the effects of DON is to cause cytotoxicity through cell apoptosis (34). To determine if the DON mimotope peptide was cytotoxic, synthetic peptide C430 (0, 0.34, 3.4, and 34 µM) was compared with DON (0 and 3.4 µM) in order to determine the cytotoxic effects of these compounds to bone marrow cells. As expected, DON at a concentration of 3.4 µM caused 40 to 60% cell death after 18 h of incubation. Synthetic peptide C430, however, did not have any adverse effect on the viability of bone marrow cells in culture at C430 concentrations up to 34 µM. When combined with DON, C430 at any of the concentrations tested did not significantly increase or decrease the cell viability resulting from the presence of DON, suggesting that there was no significant synergism or antagonism between the DON mimotope peptide and DON with respect to bone marrow cell death.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Effects of DON (3.4 µM) and synthetic peptide C430 (3.4 µM) on protein synthesis in vitro with rabbit reticulocyte lysate. The translation template was gamma globulin mRNA. The plus and minus signs indicate whether reagents were added.

Structural model of the DON mimotope peptide and comparison with the structure of DON. Within the set of nonhomologous protein structures, 31 analogs of DONPEP.2 tetrapeptides were found. The sequence search was limited to four residues, because close sequence matches to more than four residues are rarely found in the Brookhaven PDB. The secondary structures of these tetrapeptides, which were assigned based on close backbone superposition with ideal secondary structures (-helices, -strands, reverse turns), were predominately in the -strand conformation (Table 1).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In a search for DON mimotope peptides, we screened a phage-displayed random heptapeptide library and identified two closely related peptide sequences, DONPEP.2 (SWGPFPF) and DONPEP.12 (SWGPLPF), that specifically bind with high affinity to anti-DON mAb 6F5. To our knowledge, this is the first time that mimotope peptides for a nonproteinaceous, low-molecular-weight mycotoxin have been found.

In a competitive ELISA, DON could compete with selected peptides for binding to mAb 6F5, while the synthetic peptide also competed with DON for binding to the same antibody, which strongly suggested that these clones bind to the same antigen binding site of the mAb. The fact that these peptides can be used in ELISA has several implications. From a safety standpoint, it is advantageous to use a nontoxic peptide as an immunochemical reagent which replaces the toxic compound DON in an immunoassay. Second, conjugation of a peptide to peroxidase or other proteins is much easier than conjugation of DON to the same enzyme. Finally, if the peptide is genetically fused with a reporter enzyme, such as AP, chemical conjugation can be avoided. In practice, commercial ELISA for DON detect a lower range of concentrations (0.5 to 1 µg/ml; 1.7 to 3.4 µM).

Although many mimotope peptides for protein antigens have been isolated from phage-displayed peptide libraries, there have been only a few cases in which peptides have been selected as mimics of nonproteinaceous chemicals (21, 38). It is interesting to compare the peptide sequences selected with mAb 6F5 to peptide sequences that are known to interact with carbohydrate binding sites in order to identify whether there are general features common to the sequences. Our sequences were similar to the sequences of the carbohydrate mimics in that there was a clear preference for aromatic groups and proline residues (21, 38). This is not unreasonable since these amino acids resemble sugar moieties in size and cyclic shape. Furthermore, the proline residues may serve an important structural role by orienting the aromatic side chains spatially in a manner similar to the orientation of the branched carbohydrates which they mimic.

Although the peptide may have alternative conformations, the prevalent structure in the peptide analogs is an inverted-U-shaped turn. The absence of any superpositions in which nivalenol aligned predominately with one of the aromatic side chains suggests that the side chains of the peptide may not be the major epitopes recognized by mAb 6F5. Although in several of the superposition results Trp side chain atoms of SWGPFPF corresponded to nivalenol atoms, the peptide backbone, especially in the region of the first proline residue, forms the most DON-like structure in the peptide.

If a small peptide completely mimics the epitope of a certain antigen, when the peptide is conjugated on a suitable carrier protein or displayed on a phage, it can elicit antibodies directed against the native antigen whose epitope the peptide represents (2, 27, 30). Although our selected peptides and DON competed with each other for binding to mAB 6F5 and C430 can antagonize the inhibitory effects of DON in an in vitro protein synthesis system, no specific antibodies for DON were produced from C430-BSA conjugate-immunized animals when high-affinity antibodies to the peptide were detected in these animals. It is not clear whether the selected peptides mimic the surface structure of DON in some respects or bind to the cleft by an entirely different mechanism. The latter possibility was described in the case of streptavidin-binding peptides discovered with a phage-displayed library (43), in which the peptides associated with the binding site in a fashion that was quite different than the fashion used by the natural ligand, biotin. Another possibility is that there may be more than one epitope in the peptide and that the moiety which mimics DON has poor immunogenicity. This proposed mechanism is supported by the observation that a specific antibody for DON was rarely detected in large numbers of animals immunized with DON-carrier protein conjugates (6).

The toxic effects of trichothecenes are presumably related to the inhibition of protein synthesis by these compounds. The inhibitory activities of trichothecenes are due to their ability to bind to the 60S subunits of eukaryotic ribosomes and to interfere with peptidyltransferase activity (4, 37, 42). Presumably, peptides are agonists only if they undergo the same specific contacts and interactions with the ribosomes as DON. The mimotope peptides selected from the phage-displayed library in this study, however, exhibited antagonism to DON-induced protein synthesis inhibition. This suggested that the synthetic peptide might bind to the same site on a ribosome as DON but in an orientation that does not mimic the function of DON. It is notable that C430 did not potentiate or attenuate DON in causing cell death in a cell culture model. This may have been due to the fact that it was difficult for the peptide to cross the cell membrane (17). DON, which has a four-fold-lower molecule weight and a specific structure, may be more readily absorbed into cells (44).

In addition to the toxic effects of trichothecenes on animals and humans, there is a growing body of evidence which indicates that these compounds, including DON, can enhance the virulence of plant-pathogenic Fusarium species on plant hosts (36, 46). The trichothecene-producing Fusarium fungi cause a broad range of plant diseases, including head and seedling blight of small grains, such as wheat and rye, ear and stalk rot of maize, stem rot of carnation, and seedling blight and root rot of a number of other plant species, including beans, clover, and tomato (11). If the fungal virulence associated with DON is mediated through protein synthesis inhibition and the mimotope peptides described here are antagonistic to DON activity in plants, transgenic plants expressing the peptides (possibly fused to another protein) might exhibit reduced levels of wheat head scab and other plant diseases caused by the DON-producing pathogenic Fusarium species.

In summary, we identified two closely related mimotope peptides for DON and demonstrated the potential for using these peptides in DON immunoassays. We also demonstrated that the mimotopes are antagonistic to DON-induced protein synthesis inhibition. The structure of DON is mimicked only indirectly by the DON mimotope peptides, and further understanding of the mode of binding of these peptides to the same antibody or ribosomes should help elucidate the mechanisms of DON toxicity and may lead to prevention of plant and animal diseases associated with this mycotoxin.