Variability in Arabinoxylan,Xylanase Activity,and Xylanase Inhibitor Levels in Hard Spring Wheat

Arabinoxylans (AX), xylanase, and xylanase inhibitors have an important role in many cereal food processing applications. The effects of genotype, growing location, and their interaction (G × L) on AX, apparent xylanase activity, and apparent xylanase inhibition activity of Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) were investigated for six hard red and six hard white spring wheat genotypes grown at three locations. Difference in total AX level among genotypes was not determined to a significant level by genotype. Instead, variability in total AX content was largely dependent on G × L. However, total AX content was significantly different between the two wheat classes. For bran xylanase activity, 25% of the variability could be attributed to G × L interaction. Moreover, there was significant difference between the bran xylanase activities in the two wheat classes. Bran TAXI activity and XIP activity were significantly different among genotypes. Genotype contributed 72% to the variability in TAXI activity and 39% in XIP. However, no significant difference was observed among the two wheat classes for TAXI or XIP activity. These results indicate that TAXI might be a stable parameter in segregating wheat genotypes with varying xylanase activity.     

Quantification of Wheat TAXI and XIP Type Xylanase Inhibitors: A Comparison of Analytical Techniques

To date, three different techniques are available for the quantification of TAXI and XIP type proteinaceous xylanase inhibitors in cereals. A first approach is based on the determination of the residual activities of xylanases (also referred to as endo‐1,4‐β‐d ‐xylanases, EC 3.2.1.8), which are specifically inhibited by these inhibitors, after incubation with sample containing the inhibitors. The other two techniques are immunoblotting and ELISA which are based on recognition of TAXI and XIP proteins by specific antibodies. TAXI, as well as XIP, are easily extracted by aqueous buffers. Hence, the large difference in their concentrations (2–10 fold higher for XIP than for TAXI in whole meal) is not caused by differences in extractability. The repeatabilities of the three techniques are comparable. The intra‐assay and inter‐assay coefficients of variation were 6–7 and 10–14%, respectively, which is in the range of values described for methods to quantify other compounds in plant and animal tissues. The three methods give comparable results, suggesting they have similar accuracies. The choice of the technique to be used will depend not only on the sensitivity and dynamic range needed, but also on its technical simplicity and the need for high‐throughput analysis.     

Xylanase inhibitors bind to nonstarch polysaccharides

This study is an in-depth investigation of the interaction between polysaccharides and the proteinaceous xylanase inhibitors, Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibitor protein (XIP), and thaumatin-like xylanase inhibitor (TLXI). The binding affinities of all three known types of xylanase inhibitors from wheat are studied by measuring the residual xylanase inhibition activity after incubation of the inhibitors in the presence of different polysaccharides, such as beta-glucans and (arabino)xylans. The binding affinities of all three xylanase inhibitors for (arabino)xylans increased with a decreasing arabinose/xylose ratio (A/X ratio). This phenomenon was observed both with water-extractable and water-unextractable (arabino)xylans. The inhibitors also interacted with different soluble and insoluble beta-glucans. None of the inhibitors tested had the ability to hydrolyze the polysaccharides investigated. The present findings contribute to the unraveling of the function of xylanase inhibitors in nature and to the prediction of the effect of added xylanases in cereal-based biotechnological processes, such as bread making and gluten-starch separation.     

Evidence for the involvement of arabinoxylan and xylanases in refrigerated dough syruping

The relationship between syruping in refrigerated doughs upon prolonged storage and different aspects of arabinoxylan (AX) hydrolysis was investigated using Triticum aestivum xylanase inhibitor (TAXI) and different xylanases in the dough formula. Dough characteristics were evaluated with strong emphasis on the AX population and its fate as a function of storage time. Selective reduction of part of the flour endogenous xylanase activity in dough by added TAXI reduced dough syruping after 12 and 20 days of storage by 50%, providing straightforward evidence for the involvement of xylanases and, thus, AX in the syruping phenomenon. Addition of xylanases with different inhibitor sensitivities [an inhibition-sensitive Bacillus subtilis xylanase (XBS(i)) as well as a noninhibited mutant (XBS(ni)) thereof] to dough confirmed the importance of xylanases in dough syruping, on one hand, and the power of wheat flour TAXI to constitute a significant barrier against xylanase-mediated dough syruping, on the other hand. Use of xylanases with different substrate selectivities [an Aspergillus aculeatusxylanase (XAA) versus XBS(ni)] showed degradation of water-extractable AX (WE-AX) and solubilized AX to low molecular weight molecules rather than the conversion of water-unextractable AX (WU-AX) to high molecular weight water extractable components to be the main factor influencing dough syruping.     

Insight into the distribution of arabinoxylans, endoxylanases, and endoxylanase inhibitors in industrial wheat roller mill streams

To gain insight into the distribution of arabinoxylans (AX), endoxylanases, and endoxylanase inhibitors in industrial wheat roller milling, all streams, that is, 54 flour fractions, 4 bran fractions, and the germ, were analyzed for ash, starch, and protein contents, alpha-amylase activity levels, total (TOT-AX) and water-extractable arabinoxylan (WE-AX) contents, endoxylanase activity levels, and endoxylanase inhibitor (TAXI and XIP) contents. In general, bran fractions were significantly richer in TOT-AX and WE-AX contents, endoxylanase activity levels, and endoxylanase inhibitor contents than germ and, even more so, than flour fractions. In the 54 different flour fractions, minimal and maximal values for TOT-AX and WE-AX contents differed by ca. 2-fold, whereas they differed by ca. 15-fold for endoxylanase activity levels. The latter were positively correlated with ash and negatively correlated with starch content, suggesting that the endoxylanase activity in flour is strongly influenced by the level of bran contamination. TAXI contents in the flour fractions varied ca. 4-fold and were strongly correlated with bran-related parameters such as ash content and enzyme activity levels, whereas XIP contents varied ca. 3-fold and were not correlated with any of the parameters measured in this study. The results can be valuable in blending and optimizing wheat flour fractions to obtain flours with specific technological and nutritional benefits.     

Development of ELISA and immunochromatographic assay for the detection of gentamicin

Competitive direct enzyme-linked immunosorbent assay (ELISA) and the immunochromatographic assay were developed using a monoclonal antibody to detect gentamicin in the animal plasma and milk. No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA, indicating that the antibody is highly specific for gentamicin. On the basis of the standard curves, the detection limits were determined to be 0.9 ng/mL in phosphate-buffered saline (PBS), 1.0 ng/mL in plasma, and 0.5 ng/mL in milk, respectively. Recoveries of gentamicin from spiked plasma and milk at levels of 25-100 ng/mL ranged from 85 to 112%. The concentration of intramuscularly injected gentamicin was successfully monitored in the rabbit plasma through competitive direct ELISA. The detection limits were estimated to be about 6 ng/mL of gentamicin in PBS, plasma, and milk using the colloidal gold-based immunochromatographic assay, which is suitable for the simple screening of gentamicin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could complement each other as well as veterinary field and laboratory findings.     

Enzyme-linked immunosorbent assay for microcystins in blue-green algal blooms

A direct competitive enzyme-linked immunosorbent assay (ELISA) for the freshwater blue-green algal toxin microcystin (MCYST) in algae and water was developed. The assay involves coating anti-MCYST-variant leucine-arginine (LR) antibody to the ELISA plate and the use of MCYST-LR-peroxidase as the enzyme marker. The linear portion of the standard curve for MCYST in phosphate buffer containing saline (PBS) was 0.5-10.0 ng/mL (25-500 pg/assay). The minimum detection level for MCYST-LR was 0.20 ng/mL (10 pg/assay). Contaminated water could be directly used in the ELISA. The overall analytical recoveries for MCYST-LR added to water at levels of 1-20 ng/mL was 83.4%. For analysis of cellular MCYST, the toxin was first extracted from the algae with 0.1M ammonium bicarbonate, diluted with PBS to less than 0.5 mg dried algae/mL (less than 5.0 mg wet weight/mL) and directly used in the ELISA. C-18 reverse-phase Sep-Pak cartridges effectively adsorbed MCYST from the toxin-containing solutions. The toxin could be recovered from the cartridge by eluting with 60% methanol. Using this approach, an algae extract that was relatively free of MCYST was prepared and was used in a recovery study. The overall analytical recovery of MCYST added to the algae extract in the range of 0.25-20 ppm was 83% with a coefficient of variation of 11.9%. The detection limit for MCYST in dried algae was about 0.25-0.5 microgram/g (0.25-0.5 ppm) lyophilized algae sample. This method was applied for the analysis of several naturally occurring algal blooms. Limited samples were also analyzed for MYCST by liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative determination of dietary lectin activities by enzyme-linked immunosorbent assay using specific glycoproteins immobilized on microtiter plates

An immunoenzymatic method for the quantitative determination of dietary lectin activities employing immobilized glycoproteins was studied. Lectins from wheat germ (WGA), peanut (PNA), and jack bean (ConA) were added to microtiter plates coated with ovalbumin or asialofetuin and quantified by enzyme-linked immunosorbent assay (ELISA) with lectin-specific antibodies. ELISA responses for lectin activity were dose-dependent in the concentration range 30-1000 ng/mL for WGA and 80-1000 ng/mL for both PNA and ConA. Inhibition assays carried out with different saccharides confirmed that the binding of lectins to immobilized glycoproteins was specific. The proposed method is specific and sensitive, allowing the quantitative determination of lectin activities on raw samples by simple dilution of the extracts. Examples of application to wheat germ and roasted peanut extracts are reported.     

Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M1 in milk

Using a highly specific antibody against aflatoxin M1, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation of M1 in milk. RIA was sensitive in the range of 5-50 ng per assay but was subject to interference by whole milk. Extraction and cleanup were therefore necessary for the detection of M1 in milk at 0.5 ng/mL. An ELISA procedure was developed by using an aflatoxin M1-carboxymethyl-horseradish peroxidase conjugate as the ligand. Competitive assays revealed that this system was relatively more sensitive for M1 than for B1, and had a much lower degree of cross-reactivity for aflatoxins B2, G1, G2, B2a, and aflatoxicol. As low as 0.25 ng M1/mL in artificially contaminated milk (raw, whole, skim) could be detected by ELISA in 3 h without extraction or cleanup. Because of its simplicity, sensitivity, and specificity, ELISA is the preferred method for monitoring aflatoxin M1 in milk.     

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.     

Development of a Field Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of α-Amylase in Preharvest-Sprouted Wheat

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for detection of α-amylase in preharvest sprouted wheat and adapted to rapid field-use formats requiring 15–20 min to perform. Polyclonal and monoclonal antibodies were prepared to detect a mixture of high and low pI isozymes of α-amylase and high pI isozymes only. All antibodies detected α-amylase on immunoblots of either a crude wheat extract or of purified enzyme, but only the polyclonal antibodies functioned in a sandwich ELISA. Depending on the antibody combination, the tube ELISA detected either the high and low pI isozymes of α-amylase or the high pI isozymes only with a detection limit of ≈0.5–1.0 ng/mL of amylase. Wheats with falling numbers (FN) of <350 sec could be discriminated from sound wheats, with decreasing FN producing increasing assay color. Using 130 wheat grain samples, ELISA absorbances for detection of both high and low pI isozymes and of high pI isozymes only were highly positively correlated with amylase enzyme activity and negatively correlated with FN. The correlations were similar for detection of both isozyme families and for detection of high pI isozymes only. Analyses of three sets of wheat samples from different environments demonstrated that the relationship between ELISA absorbance and FN had little dependence on wheat cultivar. The precision of sample analysis using the field ELISA was similar to the precision of FN test apparatus.     

Development and fast screening of salbutamol residues in swine serum by an enzyme-linked immunosorbent assay in Taiwan

The analysis of salbutamol in swine serum is the more practical basis for large scale surveillance programs in Taiwan. Objectives of the study were to develop a new assay and to compare with a commercially available kit in field test screens. A simple and reliable enzyme-linked immunosorbent assay (ELISA) to monitor the presence of beta-agonist, salbutamol, in 1,358 field samples of swine serum that were collected from local meat markets was described. The method proved to be suitable and sensitive for the detection of beta-agonist residues caused by growth promoting dosage. The limit of detection of the developed ELISA directly performed on diluted serum was 0.25 ng/mL. The application and the results of two ELISA kits (homemade and commercially available) for the screening of salbutamol were presented. For further confirmation, all samples that showed to be ELISA positive for salbutamol residues were analyzed by GC-MS. Adopting 1 ng/mL salbutamol as a cutoff value, the commercial beta-agonist ELISA had a sensitivity of 89.2% and a specificity of 86.7% versus GC-MS at a cutoff of 1 ng/mL. The homemade salbutamol ELISA had a sensitivity of 81.1% and a specificity of 98.6% and gave a low proportion of false-positive rate results. The reliability of the developed kit in terms of the percentage of false-positive rate results is evaluated. In conclusion, a sensitive, specific salbutamol ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.     

Immunoaffinity purification of chlorimuron-ethyl from soil extracts prior to quantitation by enzyme-linked immunosorbent assay

A competitive-indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantify chlorimuron-ethyl in soil. The linear working range of the assay was from 1 to 1000 ng mL(-)(1). The assay had an I(50) value of 54 ng mL(-)(1), with a limit of detection of 2 ng mL(-)(1) and a limit of quantification of 27 ng mL(-)(1). Three soils were extracted using a carbonate buffer (pH 9.0) and the extracts spiked with chlorimuron-ethyl. Because of the effects of coextractants (matrix effects) from soil on the accuracy and precision of the ELISA, immunoaffinity chromatography (IAC) was used to purify chlorimuron-ethyl from soil extracts prior to analysis. The immunoaffinity columns, which had a total binding capacity of 1350 ng of chlorimuron-ethyl mL(-)(1) of immunosorbent, were prepared by binding anti-chlorimuron-ethyl antibodies to protein G Sepharose 4B. Although the matrix effects were largely removed using the affinity column, they could be completely removed by first passing the extract through a column containing epoxy-coupled 1,6-diaminohexane (EAH) Sepharose 4B to remove organic acids prior to IAC. Assay sensitivity was increased 100-fold using IAC to purify and simultaneously concentrate chlorimuron-ethyl from soil extracts. The purification strategy (EAH followed by IAC chromatography) removed matrix effects from all three soils and allowed for the accurate quantitation of chlorimuron-ethyl in soil extracts.     

Hapten synthesis and development of polyclonal antibody-based multi-sulfonamide immunoassays

This paper reports the synthesis of five sulfonamide derivatives, the production of broad-specificity polyclonal antibodies for immunoassay of sulfonamides, and the analysis of milk samples by developed assay. The three-step synthesis procedure reported in most of the literature was adopted and modified in this study. In the procedure, the purification of the intermediate was avoided and the time of synthesis was shortened from >20 to 6-9 h with improved yields. This method is generally applicable to the synthesis of haptens containing the common structure of sulfonamides. Three haptens were coupled to keyhole limpet hemocyanin, and polyclonal antibodies were obtained from rabbits immunized with these conjugates. Using the antibodies obtained, from one of these was developed an enzyme-linked immunosorbent assay (ELISA) based on the competition between free sulfonamides and the hapten-horseradish peroxidase (HRP) conjugates. The hapten-HRP conjugate giving the best competitive results and 11 structurally different sulfonamides showed 50% inhibition at concentrations of <100 ng mL(-1). After removal of the protein with acetone, milk samples were analyzed by ELISA directly; a matrix effect could be avoided when a 1:20 dilution with phosphate-buffered saline was used, and 104-131% recoveries of spiked samples were obtained. The developed immunoassay is suitable to determine sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine, sulfapyridine, and sulfamethizole below the maximum residue limit in milk (100 ng mL(-1) of total sulfonamides) rapidly and reliably.     

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.     

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.     

Xylanase,beta-glucanase,and other side enzymatic activities have greater effects on the viscosity of several feedstuffs than xylanase and beta-glucanase used alone or in combination

This study was carried out to evaluate the effects of a pure xylanase, a pure beta-glucanase, a mix of the two pure enzymes, and a commercial enzyme preparation (Quatrazyme HP, Nutri-Tomen Les Ulis, France) on the viscosity exhibited by water-soluble nonstarch polysaccharides of several feedstuffs (Rialto wheat, Sidéral wheat, Isengrain wheat, triticale, rye, barley, oats, corn, wheat bran, rice bran, wheat screenings, soybean meal, rapeseed meal, sunflower meal, and peas). The viscosity depended on the feedstuffs and varieties of the same feedstuff. There was a correlation (R (2) = 0.86) between viscosity of cereals and their arabinoxylan and beta-glucan contents. The correlation was greater (R (2) = 0.99) when the type of cereal was taken into account. The addition of pure xylanase significantly decreased the viscosity of all feedstuffs except sunflower meal (P < or = 0.05). However, pure beta-glucanase was unable significantly to decrease the viscosity of Isengrain wheat, corn, rice bran, wheat screenings, soybean meal, and sunflower meal. There was a greater decrease in viscosity with the combination of xylanase and beta-glucanase than with addition of xylanase or beta-glucanase alone. This synergistic action of xylanase and beta-glucanase was observed only in Rialto wheat, Sidéral wheat, triticale, rye, barley, oats, and peas. Finally, the commercial enzyme preparation produced a greater reduction (P < or = 0.05) in viscosity for all feedstuffs compared to xylanase or beta-glucanase used alone or in combination. The greater effectiveness of the commercial enzyme preparation was due to the presence of side enzymatic activities (arabinofuranosidase, xylosidase, glucosidase, galactosidase, cellulase, and polygalacturonase).     

Impact of inhibition sensitivity on endoxylanase functionality in wheat flour breadmaking

A Bacillus subtilis endoxylanase (XBS(i)) sensitive to inhibition by Triticum aestivum L. endoxylanase inhibitor (TAXI) and a mutant thereof (XBS(ni)), uninhibited by TAXI, were used in straight-dough breadmaking to assess the importance of endoxylanase inhibition sensitivity on endoxylanase functionality in the process. With two European wheat flours, the loaf volume improving effect of XBS(ni) at much lower enzyme dosages was substantially larger than that brought about by XBS(i). This coincided with differences in arabinoxylan (AX) hydrolysis. Although XBS(ni) had a lower substrate selectivity for water-unextractable arabinoxylan (WU-AX) than XBS(i), the former solubilized significantly more WU-AX than XBS(i). Because of inhibition, XBS(i) solubilized most of the WU-AX during mixing, whereas, with XBS(ni), the rate of solubilization decreased less with increasing processing time than that with XBS(i). During fermentation and baking and at the highest dosage (600 U/kg of flour of XBS(i) and 60 U/kg of flour of XBS(ni)), XBS(ni) induced a stronger degradation of enzymically solubilized and water-extractable AX than XBS(i). Taken together, the data clearly demonstrate that endoxylanases, which in vitro are inhibited by endoxylanase inhibitors and still are active in the breadmaking process, as demonstrated by their functional (bread volume) enhancing effect, gradually lose their activity in the process.     

Enzyme-linked immunosorbent assay based on a polyclonal antibody for the detection of the insecticide fenitrothion. Evaluation of antiserum and application to the analysis of water samples.

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the organophosphorus insecticide fenitrothion, the specificity of the antiserum R-3 generated with the bifunctional hapten, LysMNPA (2-[[[(3-methyl-4-nitrophenyl)oxy]methylcarbonyl]amino]-6-(2,4-dinitrophenyl)aminohexanoic acid) and the application to the residual analysis of some water samples were evaluated. At optimized ELISA conditions, the quantitative working range was from 1 to 39 ng/mL with a limit of detection of 0.3 ng/mL and an IC(50) value of 6 ng/mL. Cross-reactivity to structurally similar organophosphorus compounds and related chemicals was determined. The antiserum R-3 showed significant cross-reactivity with fenitrooxon and 3-methyl-4-nitrophenol, which have a 3-methyl-4-nitrophenoxy group as common structures, but showed relatively low cross-reactivity with other compounds. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween 20 in the assay buffer. These four kinds of water samples were fortified with fenitrothion at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution. The mean recovery was 105.9%, and the mean coefficient of variation was 10.9%. The results suggested that the developed ELISA would be very suitable for a preliminary screening for fenitrothion in water samples at such low levels.     

TAXI type endoxylanase inhibitors in different cereals

An affinity-based purification procedure with the immobilized family 11 Bacillus subtilis endoxylanase XynA allowed us to obtain high yields of highly pure endoxylanase inhibitor fractions from rye, barley, and durum wheat. In contrast, no inhibitors interacting with the B. subtilis endoxylanase affinity column are present in corn, buckwheat, rice, and oats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and inhibitor specificity showed that the isolated inhibitors belonged to the TAXI endoxylanase inhibitor family, thus providing a view on the diversity of this cereal inhibitor family. The isolated inhibitors are basic proteins of ca. 40 kDa, occurring in two molecular forms, with pI values of ca. 8.5 (durum wheat) and ca. 9.0 (rye, barley). They are, in general, strong inhibitors of family 11 endoxylanases but not of family 10 endoxylanases. Because cereal endogenous endoxylanases belong to the latter family, this probably indicates that they do not influence cereal metabolic processes. For the first time, endoxylanase inhibitors, similar to TAXI I and TAXI II from wheat, were isolated from durum wheat and characterized. For each cereal, high-resolution cation exchange chromatography revealed the presence of multiple isoinhibitors, each of which occurs in two molecular forms. However, in durum wheat and barley, a single isoform is predominantly present.