Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges.

A lateral-flow immunoassay using a colloidal gold-labelled monoclonal antibody was developed for the rapid detection of deoxynivalenol (DON). Different parameters, such as the amount of immunoreagents, type of the materials, composition of the blocking solution and of the detector reagent mixture, were investigated to provide the optimum assay performance. The experimental results demonstrated that such a visual test had an indicator range rather than a cut-off value. Thus, tests for DON determination with two different indicator ranges of 250-500 and 1000-2000 microg kg(-1) were designed. The method allowed detection of DON at low and high concentration levels, which could be useful for research and practical purposes. The assay applied to spiked wheat and pig feed samples demonstrated accurate and reproducible results. The applicability of the developed lateral-flow test was also confirmed under real field conditions. The test strips prepared in Belgium were sent to Mexico, where they were used for the screening of DON contamination in different bread wheat entries from Fusarium Head Blight inoculated plots. The results were compared with those obtained by ELISA and LC-MS/MS. A poor correlation between ELISA and LC-MS/MS was observed. Visual results of the dipstick tests were in a good agreement with the results of the LC-MS/MS method. Coupled with a simple and fast sample preparation, this qualitative one-step test based on the visual evaluation of results did not require any equipment. Results could be obtained within 10 min. The described assay format can be used as a simple, rapid, cost-effective and robust on-site screening tool for mycotoxin contamination in different agricultural commodities.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of zearalenone and deoxynivalenol.

A multianalyte lateral-flow technique using colloidal gold-labeled monoclonal antibodies was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEA). The results of this qualitative one-step test were interpreted visually. A very simple and fast sample preparation was used, and the assay procedure could be accomplished within 10 min. When applied to spiked wheat samples, the technique gave accurate and reproducible results. Cut-off levels of 1500 and 100 microg kg(-1) for DON and ZEA, respectively, were observed. The described multianalyte format can be used as a reliable, rapid and cost-effective on-site screening technique for the simultaneous determination of mycotoxins in grain samples.

Application and validation of a clean-up tandem assay column for screening ochratoxin A in cocoa powder.

A rapid antibody-based assay for the detection of ochratoxin A in cocoa powder is described, involving sequential clean-up and visual detection of the toxin ("clean-up tandem assay column"). The screening test was developed to have a cut-off level of 2 microg kg(-1) and was shown to have false positive and false negative rates of 10 and 2%, respectively. Analysis of six samples can be carried out in the field in approximately 30 min by untrained workers. Using the proposed rapid screening test, 10 retail cocoa powders were found to contain no detectable levels of ochratoxin A (<2 microg kg(-1)). These samples were also found to be negative (<2 microg kg(-1)) when analysed using an LC-MS/MS method.

Evaluation of fumonisin contamination in cornflakes on the Belgian market by "flow-through" assay screening and LC-MS/MS analyses.

A total of 205 cornflake samples collected in Belgian retail stores during 2003-2004 were surveyed for the natural occurrence of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3). These cornflake samples, originating from conventional as well as from organic production, were analyzed using an intralaboratory-validated LC-MS/MS method. Additionally, 90 cornflake samples were subjected to rapid screening using a flow-through enzyme immunoassay method to demonstrate the practicability of a screening test coupled to a validated confirmatory LC-MS/MS method for the management of food safety risks. FB(1) concentrations ranged from not detected (nd) [LOD (FB1) = 20 microg/kg] to 464 microg/kg with mean and median concentrations of respectively 104 +/- 113 and 54 microg/kg. For FB2 and FB3, the concentration ranges varied respectively from nd [LOD (FB2) = 7.5 microg/kg] to 43 microg/kg and from nd [LOD (FB3) = 12.5 microg/kg] to 90 microg/kg. Mean concentrations for FB2 and FB3 were respectively 12 +/- 8 and 21 +/- 15 microg/kg, while the median concentration was 11 microg/kg for FB2 and 19 microg/kg for FB3. From the statistical tests (chi2 and ANOVA model III), it could be concluded that the agricultural practice did not have any significant effect on the fumonisin concentrations but that the variation between different batches was significant (p < 0.0001).

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.

Development of a solid-phase cleanup and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin a in roasted coffee.

A membrane-based flow-through enzyme immunoassay (patent application pending) for the detection of ochratoxin A (OA) in roasted coffee was developed. First, an extraction and solid-phase cleanup method was developed. A high partition coefficient for OA in the mobile phase was achieved by using methanol/5% aqueous NaHCO(3) as the sample extraction and cleanup solvent. The solid-phase (aminopropyl) cleanup was developed to chromatographically elute OA but retain cross-reacting compounds. Without using aminopropyl cleanup, cross-reacting compounds resulted in 100% false positives for both flow-through enzyme immunoassay and HPLC methods. However, after cleanup with aminopropyl, no false positives were observed. The flow-through results were visually evaluated. The sensitivity achieved for the flow-through was 4 microg kg(-1) in spiked roasted coffee. The assay was used to screen roasted coffee samples. Results were confirmed with HPLC with a detection limit of 1 microg kg(-1).

Optimization of solid-phase clean-up prior to liquid chromatographic analysis of ochratoxin A in roasted coffee.

An extraction and clean-up method for ochratoxin A (OA) in roasted coffee has been developed and the HPLC method optimized. An interfering compound with a similar retention time as OA was adsorbed by the aminopropyl (NH2) material at < or = 5% NaHCO3. Residual OA on the column was recovered by washing with the extraction solution followed with methanol. Fractions were mixed together for further clean-up with Ochratest immunoaffinity columns (IACs). Analysis by HPLC resulted in a well resolved OA peak and reduction in matrix interferences. Recoveries ranged from 72 to 84% and the detection limit was 1 ng/g.

A collaborative study to validate novel field immunoassay kits for rapid mycotoxin detection.

Kits designed to detect ochratoxin A (OA) and T-2 toxin by a membrane-based flow-through enzyme immunoassay were studied collaboratively by screening cereals (wheat, rye, maize and barley) for the presence of these mycotoxins. Sample preparation and test procedure were clearly described in the instruction leaflets included in the kits. A simple methanol-based extraction followed by filtration and dilution steps was prescribed. Reagents were successively pipetted to the membrane of the device, then colour development was evaluated visually. Limits of detection for the ochratoxin A and T-2 toxin tests were 4 and 50 microg kg(-1), respectively. Five laboratories took part in the first stage of this study, and five more joined the second stage. Cereal samples (blank, spiked or inoculated) were shipped with the kits to the participating laboratories, while results obtained were confirmed by high-performance liquid chromatography with fluorescence detection and by gas chromatography-mass spectrometry for ochratoxin A and T-2 toxin, respectively. Some initial difficulties were encountered. In the second stage, four ochratoxin A and four T-2 toxin kits were used by 10 collaborators to analyse 21 cereal samples. For the ochratoxin A kits, the percentage of false positive and false negative results were 2% and 4%, respectively. The results of one T-2 toxin kit were outliers and when excluded, the overall percentage false positive and false negative results were 6% and 3%, respectively.