Development of surface plasmon resonance-based immunoassay for aflatoxin B(1)

Aflatoxins are a group of highly toxic fungal secondary metabolites that occur in Aspergillus species and may contaminate foodstuffs and feeds. Two different anti-aflatoxin B(1) antibodies were examined to develop a surface plasmon resonance (SPR)-based immunoassay to aflatoxin B(1). A conjugate consisting of aflatoxin B(1)-bovine serum albumin (BSA) was immobilized on the dextran gel surface. Competition between immobilized aflatoxin B(1) conjugate and free aflatoxin B(1) in solution for binding to antibody injected over the surface formed the basis for the assay. Regeneration of the antibody from the immobilized conjugate surface is essential for the development of such an inhibitive immunoassay. Problems were encountered with the regeneration of the sensor surface, due to the high-affinity binding of the antibodies. Conventional regeneration solutions consisting of low concentrations of NaOH and HCl worked to a degree, but regeneration was at the expense of the integrity of the immobilized conjugate. A polyclonal anti-aflatoxin B(1) antibody was produced and was found to be regenerable using an organic solution consisting of 1 M ethanolamine with 20% (v/v) acetonitrile, pH 12.0. This combined high ionic strength and extreme pH, as well as chaotrophic properties and allowed the development of an inhibitive immunoassay. The assay had a linear range of 3.0-98.0 ng mL(-1) with good reproducibility.     

Rapid fluorescence polarization immunoassay for the mycotoxin deoxynivalenol in wheat

The fungus Fusarium graminearum, a pathogen of both wheat and maize, produces a toxin, deoxynivalenol (DON), that causes disease in livestock. A rapid test for DON in wheat was developed using the principle of fluorescence polarization (FP) immunoassay. The assay was based on the competition between DON and a novel DON-fluorescein tracer (DON-FL2) for a DON-specific monoclonal antibody in solution. The method, which is a substantial improvement over our previous DON FP immunoassay, combined a rapid (3 min) extraction step with a rapid (2 min) detection step. A series of naturally contaminated wheat and maize samples were analyzed by both FP immunoassay and liquid chromatography (HPLC-UV). For wheat the HPLC-UV and FP methods agreed well (linear regression r(2) = 0.936), but for maize the two methods did not (r (2) = 0.849). We conclude that the FP method is useful for screening wheat, but not maize, for DON.     

Comparative sensitivity of immunoassays for haptens using monomeric and dimeric antibody fragments

A single-chain anti-atrazine antibody fragment, scAb (single-chain Fv with a CK domain), was expressed in Escherichia coli, and monomeric and dimeric species were preferentially purified from periplasmic extracts by chromatography upon nickel chelate immunosorbent columns or by immunoaffinity purification using a constant domain (CK) tag. Recombinant monomeric and dimeric antibody fragments, Fab, and intact monoclonal antibodies were compared in assays by competition between free atrazine in solution and (a) immobilized atrazine-bovine serum albumin conjugate (indirect assay) or (b) atrazine-alkaline phosphatase (direct assay). Recombinant antibody fragments provided a lower detection limit than either Fab or intact monoclonal antibody in both assay formats. Monomeric fragments displayed a sensitivity of detection down to 0.1 ppb, compared to 1.0 ppb for dimeric fragments and the parental monoclonal.     

Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat

Novel qualitative as well as semiquantitative rapid strip tests for screening of T-2 mycotoxin in agricultural commodities were developed. Colloidal gold particles were coated with monoclonal anti-T-2 antibodies and used as detector reagent, indicating the strip test results by formation of up to two colored lines in a competitive assay format. The test line comprises a protein conjugate of the T-2 mycotoxin and the control line an antispecies-specific antibody to confirm the correct test development. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with methanol/water (70:30) by shaking for 3 min and the extract directly used without further cleanup steps. The T-2 toxin lateral flow device (LFD) presented has a cutoff level around 100 microg/kg for naturally contaminated wheat and oat. The semiquantitative test may be used in the lower micrograms per kilogram range and allows for rapid semiquantitative photometric classification of the level of sample contamination. For both tests, results were obtained within 4 min. The developed LFDs therefore allow for the first time fast and on-site screening for the determination of T-2 toxin in cereals.     

Enzyme-linked immunosorbent assay of aflatoxins B1, B2, and G1 in corn, cottonseed, peanuts, peanut butter, and poultry feed: collaborative study

A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of less than 30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain greater than or equal to 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain less than 20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing less than 2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and greater than or equal to 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 10:1:3) were 52, 86, and 96%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed

The aim of this work was to develop an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B(1) in pig feed. The test consisted of three main components: conjugate pad, membrane, and absorbent pad. The membrane was coated with two capture reagents, that is, aflatoxin B(1)-bovine serum albumin conjugate and rabbit anti-mouse antibodies. The detector reagent consisted of colloidal gold particles coated with affinity-purified monoclonal anti-aflatoxin B(1) antibodies, which saturated the conjugate pad. A comparison of several extraction methods for the pig feed matrix is presented. A mixture of methanol/water (80:20, v/v) gave the best recoveries. After sample extraction and dilution, the dipstick was put in the sample solution at the conjugate pad side and developed for 10 min. Analyte present in the sample competed with the aflatoxin B(1) immobilized on the membrane for binding to the limited amount of antibodies in the detector reagent. Thus, the line color intensity of an aflatoxin B(1)-positive dipstick is visually distinguishable from that of an aflatoxin B(1)-negative sample. The visual detection limit for aflatoxin B(1) is 5 microg/kg. The major advantages of this one-step striptest are that results can be obtained within 10 min and that all reagents are immobilized on the lateral flow dipstick.     

Solvolysis procedures for the determination of bound residues of the mycotoxin deoxynivalenol in fusarium species infected grain of two winter wheat cultivars preinfected with barley yellow dwarf virus

A trichloroacetic acid treatment at 140 degrees C for 40 min was successfully established as a suitable solvolysis procedure for the recovery of bound deoxynivalenol or its derivatives in Fusarium-infected plants. Deoxynivalenol itself was not decomposed in the procedure. The derivative 15-acetyl-deoxynivalenol was chosen as a model compound for setting free deoxynivalenol in an acid-catalyzed deesterification reaction, developing the method. This is the first report using a trichloroacetic acid solvolysis procedure as a sample incubation step to free bound deoxynivalenol and determine free from bound deoxynivalenol in the sample. Between 13 and 63% of the total deoxynivalenol consisted of nonextractable deoxynivalenol. Deoxynivalenol contents in grain of the susceptible cultivar "Agent" infected with Fusarium spp. were 12-24 times higher when compared to those for the corresponding moderately resistant cultivar "Petrus". The highest deoxynivalenol amounts were determined in grain infected with Fusarium spp. as well as simultaneously infected with BYDV. This solvolysis procedure may be of importance for distinguishing between resistant and susceptible plants and their ability to immobilize (bound) mycotoxins as a plant defense mechanism.     

Use of phosphonic acid as a generic hapten in the production of broad specificity anti-organophosphate pesticide antibody

Phosphonic acid (trans-4-phosphono-2-butenic acid; TPB) was used as a generic hapten in order to generate broad specificity antibodies against a group of organophosphorus pesticides. The polyclonal antiserum showed, in an indirect enzyme-linked immunosorbent assay (ELISA) format, preferential binding toward pesticides containing unsaturated diethyl-phosphate functionalities rather than the equivalent thiophosphate or dimethyl structures. The level of detection in the ELISA using a heterologous system was investigated and showed a 20-fold improvement when a conjugate for which the antibody had lower affinity was immobilized on the plate. Biosensor assays using parathion as a standard indicated that the antibody had a relatively high dissociation rate, and reproducible cycles of regeneration were achieved. The potential for using TPB as a generic hapten is discussed.     

Deoxynivalenol removal from barley intended as swine feed through the use of an abrasive pearling procedure

Samples of naturally contaminated hulled barley, with varying deoxynivalenol concentrations, were subjected to an abrasive type dehulling procedure. The remaining grain fractions were analyzed for weight remaining (%), deoxynivalenol (ppm), crude protein (%CP), neutral detergent fiber (%NDF), ash (%ASH), gross energy (GE; kcal/kg), and calculated digestible energy values (DE; kcal/kg). Following the initial 15 s of pearling, 85% of the grain mass remained. Additional pearling resulted in a linear decline of grain mass. Following 15 s of pearling, the grain contained 34% of the initial deoxynivalenol content, irrespective of the initial level of contamination. Further pearling resulted in continued significant (p < 0.05) reductions in the percent of deoxynivalenol remaining to a level of 7.9% after 120 s but with significant losses in grain mass. Pearling can serve as an effective means of reducing the deoxynivalenol content of barley, with improvements in nutrient levels. However, the need to reduce the deoxynivalenol content of contaminated barley to less than 1 ppm for swine will necessitate the removal of a significant amount of the grain mass for heavily contaminated samples.     

Enzyme-linked immunosorbent assay for deoxynivalenol in corn and wheat

The availability of antibody against deoxynivalenol (DON) triacetate (Tri-Ac-DON) has enabled development of a direct enzyme-linked immunosorbent assay (ELISA) and an indirect ELISA for DON in corn and wheat. In both assays, DON is extracted from the sample with acetonitrile-water, reacted with acetic anhydride to form Tri-Ac-DON, and diluted in phosphate buffer for analysis. Direct ELISA was found to be the more sensitive procedure. Fewer interferences are evidenced, and the assay is less time consuming than is indirect ELISA. For direct ELISA, recovery of 10-1000 ppb DON added to corn and wheat was 100% (SD 15, CV 15%) and 102.1% (SD 12.2, CV 11.9%), respectively. For indirect ELISA, overall recovery of 10-1000 ppb DON added to wheat was 121.5% (SD 39.5, CV 32.5%); in the higher concentration range (500-1000 ppb), recovery was 105% (SD 18, CV 17%). The minimal detection level for DON was around 10 ppb. Analysis of 7 naturally contaminated samples for DON showed that the ELISA results agreed well with those obtained by radioimmunoassay and thin-layer chromatography.     

Gas chromatographic determination of deoxynivalenol in wheat with electron capture detection: collaborative study

Ten laboratories participated in a collaborative study of a method for the determination of deoxynivalenol in wheat by gas chromatography with electron capture detection. Each laboratory analyzed 6 samples in duplicate. Each collaborator received samples spiked at the 100.3, 501.3, and 1002.6 ng/g levels; a control sample; and 2 naturally contaminated samples. The average recovery (outliers excluded) for the spiked samples was 92.2%. The mean repeatability and reproducibility, respectively, were 32.2 and 41.3% for the spiked samples and 30.9 and 47.6% for the naturally contaminated samples. The method was adopted official first action.     

Assessment of extraction procedures in the analysis of naturally contaminated grain products for deoxynivalenol (vomitoxin)

A comparison of 2 extraction solvent systems (acetonitrile-water, 21 + 4 and methanol-water, 1 + 1) and 3 mixing apparatus (high-speed blender, wrist-action shaker, and mechanical stirrer) was carried out for different extraction time periods. Methods were evaluated using uncontaminated corn spiked with pure deoxynivalenol (DON), field-inoculated (Fusarium graminearum) corn, and uncontaminated and naturally infected wheat in swine diets. After sample extraction, aliquots were passed through alumina-charcoal cleanup columns, evaporated to dryness, dissolved in 8% aqueous methanol, and injected onto the liquid chromatograph. Results confirm published reports of recoveries from DON-spiked samples; however, longer extraction times (less than or equal to 120 min) were required for naturally contaminated samples. Use of the high-speed blender resulted in faster extractions, but in our laboratory more samples could be more conveniently extracted simultaneously with the wrist-action shaker or mechanical stirrer. Less carryover (co-extraction) of interfering contaminants was observed when acetonitrile-water was used vs methanol-water. Results emphasize the importance of careful evaluation of extraction procedures with not only spiked samples but also naturally contaminated samples to establish extraction times required for maximum deoxynivalenol recoveries.     

Development of a fluorescence polarization assay for the determination of aflatoxins in grains

Aflatoxins produced by Aspergillus flavus are commonly found in human and animal foods including grains, cereals, peanut products, sorghum, and soy seeds. Exposure to aflatoxins has been associated with carcinogenicity. This paper reports a simple, portable, and rapid fluorescence polarization (FP) assay for aflatoxin determination in grains. This immunoassay is field portable, homogeneous, and without any washing and cleaning steps. The assay is based upon the competition between free aflatoxin and an aflatoxin-fluorescein tracer for an aflatoxin-specific monoclonal antibody in solution. A series of naturally contaminated corn, sorghum, peanut butter, and peanut paste samples were analyzed by FP and compared with HPLC results. Similarly, spiked popcorn samples were analyzed by FP. FP results of naturally contaminated samples correlated well with HPLC (r (2) = 0.97). FP analysis of spiked popcorn samples (with a mixture of B(1)/B(2)/G(1)/G(2), 7/1/3/1, w/w) gave a good correlation with spiked values (r (2) = 0.99). However, FP consistently underestimated the aflatoxin contents. This was perhaps due to low cross-reactivity of the antibody used toward B(2), G(1), and G(2) aflatoxins. These results combined with the portability and simplicity of the assay suggest that the assay can be used for screening total aflatoxin in grains.     

Cross-reactivity of antibodies in some commercial deoxynivalenol test kits against some fusariotoxins

Cross-reactivity of antibodies in AGRAQUANT, DON EIA, VERATOX, ROSA LF-DONQ, and MYCONTROLDON designed for deoxynivalenol (DON) determination in food and feedstuffs was evaluated against nivalenol, 3-acetylDON, 15-acetylDON, de-epoxy metabolite 1 of DON, DON-3β-glucoside, T2-toxin, HT2-toxin, fusarenone X, diacetoxyscirpenol, verrucarol, and zearalenone. Cross-reactivity measurements were run in water using the 50% reduction of absorbance of the blank for ELISA kits or through direct DON determination upon using the standards of mycotoxins via ROSA LF-DONQ or MYCONTROLDON. For the tested toxin concentrations, all DON kits have low cross-reactivity toward diacetoxyscirpenol, T2-toxin, HT2-toxin, verrucarol, and zearalenone and moderate cross-reactivity toward 15-AcetylDON and fusarenone X. AGRAQUANT, DON EIA, and VERATOX kits showed high cross-reactivity in various ranking orders against DON-3-Glc, DOM-1, and 3AcDON. DON EIA showed also high cross-reactivity against nivalenol and fusarenone X. These mycotoxins could coexist in food or feedstuffs, and analytical results can be wrongly interpreted. Cross-reactivity does not allow checking the compliance with the legal norms, but it does allow an overall risk assessment for the consumers. Updating regularly the cross-reactivity evaluation of the produced batches is recommended for 3-acetylDON, nivalenol, DON-3-Glc, de-epoxy metabolite 1, and fusarenone X.     

Confirmation of reduced toxicity of deoxynivalenol in extrusion-processed corn grits by the MTT bioassay

The objective of this study was to determine the loss of toxicity of deoxynivalenol in extruded cereal-based products by the tetrazolium salt (MTT) bioassay using a sensitive Chinese hamster ovary (CHO-K1) cell line and to compare the results to chemical (high-performance liquid chromatography, HPLC) and biochemical (enzyme-linked immunosorbant assay, ELISA) methods of analysis. A split-split plot design was used for the extrusion process experiments at temperatures of 150, 175, and 200 degrees C and screw speeds of 70 and 140 rpm. The initial mean deoxynivalenol concentration in the corn grits artificially contaminated with Fusarium graminearum was found to be 23.5 mug/g as measured by HPLC. The percent reductions of deoxynivalenol in the contaminated corn grits upon extrusion processing ranged from 22 to 35%, from 21 to 34%, and from 21 to 37% as measured by HPLC, ELISA, and MTT bioassay, respectively. The MTT bioassay results were more closely correlated with HPLC (r = 0.90) results than with ELISA results (r = 0.78). The MTT bioassay, using a sensitive mammalian cell line, was demonstrated to be a useful method for quantification of deoxynivalenol as well as a potential toxicity screening method for contaminated extruded cereal-based products.     

Radioimmunoassay of deoxynivalenol in wheat and corn

With the availability of antibody against deoxynivalenol triacetate (DON-triacetate), a radioimmunoassay (RIA) for DON in wheat was developed. DON is extracted from the sample with acetonitrile-water (84 + 16), defatted with hexane, and then reacted with acetic anhydride to form DON-triacetate. The reaction mixture is loaded onto a C-18 cartridge to remove excess reagents and impurities. Acetylated DON is eluted from the cartridge with 50% methanol in water, and then analyzed by radioimmunoassay utilizing antiserum against DON-triacetate and tritiated DON-triacetate. Overall recovery for DON added to wheat between 50 and 5000 ppb was 86% with a standard deviation of 7% and coefficient of variation of 8%. The limit of detection for DON was about 20 ppb. Analysis of 12 naturally contaminated wheat, corn, and mixed feed samples for DON revealed that RIA results agreed well with thin layer chromatographic analyses performed by other laboratories.     

Surface plasmon resonance analysis on interactions of food components with a taste epithelial cell model

A new device for evaluating the continuity of taste was developed with the use of surface plasmon resonance (SPR). The model of lingual cells was constructed with liposomes immobilized onto an L1 sensor chip for SPR. Using this device, we classified food components into three categories according to the sensorgram pattern and residual ratio on lipid bilayer. Samples in group A strongly interacted with lipid bilayer, those in group B poorly interacted, and those in group C belong to neither group A nor group B. Sweet proteins and gymnemic acids that prolonged sweet perception were categorized in group A. Almost all the carbohydrates investigated and aspartame, of which the taste perception does not continue, belonged to group B. This device made it possible to detect the interaction with lipid bilayer and dissected the mechanism of taste continuity.     

Determination of ractopamine in cattle and sheep urine samples using an optical biosensor analysis: comparative study with HPLC and ELISA

A biosensor method, using the surface plasmon resonance (SPR) principle, was developed for the determination of ractopamine in cattle and sheep urine. A monoclonal antibody was used to compete with ractopamine in the sample and ractopamine immobilized on the sensor chip. Addition of bovine serum albumin (BSA, 1 mg/mL) as an antibody stabilizer to the incubation buffer was required to achieve a stable biosensor response throughout each sample set. The calibration curve gave a mean IC(50) of 4.7 +/- 0.21 ng/mL (n = 7). Over sample concentrations from 2.5 to 10 ng/mL recoveries were typically approximately 100-110%, whereas inter- and intra-assay reproducibilities (% CV) were usually less than 10 and 6%, respectively. Comparison of biosensor results with results obtained from high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assays (ELISA) using enzyme-hydrolyzed urine (to convert ractopamine conjugates to free ractopamine) gave correlation coefficients of 0.94 for sheep and 0.86 for cattle. Slopes of the lines, with zero intercepts, equaled 0.80 for sheep and 0.74 for cattle. For untreated (nonhydrolyzed) urine samples, the correlations between biosensor and HPLC results were 0.95 for sheep and 0.72 for cattle with slopes of 1.18 (sheep) and 1.69 (cattle). The slopes greater than unity indicate that the biosensor responded to ractopamine metabolites in addition to free ractopamine. The biosensor assay is an excellent analytical tool to screen ractopamine residues in sheep or cattle urine, and the results should be extendible to other species with suitable validation.     

A sensitive enzyme-linked immunosorbent assay for detecting carcinogenic aristolochic acid in herbal remedies

Aristolochic acid, a naturally occurring nephrotoxin and rodent carcinogen, has been associated with the development of various nephropathies in humans. Developing a sensitive and rapid method to screen the aristolochic acid levels in herbal remedies is urgent for protecting public health. Polyclonal antibodies for aristolochic acid were generated from rabbits after the animals had been immunized with either aristolochic acid-ovalbumin (OVA) or aristolochic acid-keyhole limpet hemocyanin (KLH). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) and a competitive direct enzyme-linked immunosorbent assay (cdELISA) were used for the characterization of the antibodies and for analysis of aristolochic acid contaminated in herbal medicine and diet pills. The antibody titers in the serum of rabbits immunized with aristolochic acid-OVA were considerably higher than those from aristolochic acid-KLH-immunized rabbits. The antibodies from the aristolochic acid-OVA-immunized rabbits were further characterized. In the ciELISA with aristolochic acid-KLH as the plate-coating antigen, the concentrations of the aristolochic acid mixture, aristolochic acid I, and aristolochic acid II that caused 50% inhibition (IC50) of binding of antibodies to aristolochic acid-KLH were found to be 1.2, 0.7, and 18 ng/mL, respectively. When 0.25-5 microg/g of standard aristolochic acid was spiked to ground lotus seeds and then extracted with 0.01 M phosphate-buffered saline, the recovery rate was found to be 86.5% in the ciELISA. Analysis of aristolochic acid in herbal medicine and diet pills with ciELISA showed that 10 of the 12 examined samples were contaminated at levels from 0.6 to 655 microg/g. The presence of aristolochic acid was also confirmed by the high-performance liquid chromatography method.     

Detection of streptomycin residues in whole milk using an optical immunobiosensor.

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples ( approximately 3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).