Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine

A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer, has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and 5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min. The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation (r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for 70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for white and rosé wines, which are known to contain less interfering compounds such as polyphenols.     

Multiplex flow-through immunoassay formats for screening of mycotoxins in a variety of food matrices

Two multi-analyte flow-through immunoassay formats for rapid detection of mycotoxins in a variety of food matrices (peanut cake, maize, and cassava flour) were developed and evaluated. The selected food matrices are typical staple foods and export products for most low-income communities around the world. The assay formats included gel-based and membrane-based flow-through assays and were based on the principle of indirect enzyme-linked immunosorbent assay. Using the same immunoreagents, the performance characteristics of both assays were compared. To the best of our knowledge, this is the first report on such a comparison. The gel-based format was developed to screen for ochratoxin A, fumonisin B₁, deoxynivalenol, and zearalenone detection at cut-off values of 3, 1,250, 1,000, and 200 μg kg⁻¹, respectively, while the membrane-based format can be used to screen ochratoxin A, aflatoxin B_1, deoxynivalenol, and zearalenone at the following cut-offs: 3, 5, 700, and 175 μg kg⁻¹, respectively. The applicability of these assay formats was demonstrated by evaluating the performance characteristics of both tests through performing multiple experiments on different days. Both assays were further evaluated by analyzing naturally contaminated samples in the laboratory and also in the field under tropical conditions (Cameroon, West Africa). The false-negative rate with both formats was less than 5%, which is in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes.     

Rapid all-in-one three-step immunoassay for non-instrumental detection of ochratoxin A in high-coloured herbs and spices

A feasible three-step method for ochratoxin A (OTA) rapid detection was developed and applied for OTA screening in high-coloured matrices such as liquorice, ginger, nutmeg, black pepper, white pepper and Capsicum spp. spices at a control level of 10 μg kg⁻¹. The method was based on the clean-up tandem immunoassay column and involved three steps: extract application, washing step and application of chromogenic substrate. A significant simplification of the assay was reached by using an additional frit with conjugate inside the clean-up tandem immunoassay column. The time for analysis was less than 10 min, including 5 min for colour development. Results were visually evaluated as colour development for negative result or no colour development for positive result. The method was coupled with a simple methanol-based extraction. A total of 27 samples were screened for OTA with the proposed method. It was shown that two samples of red pepper and one sample of liquorice, pili-pili, chilli and cayenne were contaminated with OTA above the control level at 10 μg kg⁻¹, but none of tested ginger, nutmeg, black pepper and white pepper.     

Development of colloidal gold-based flow-through and lateral-flow immunoassays for the rapid detection of the insecticide carbaryl

One-step membrane-based competitive colloidal gold-based immunoassays in flow-through and lateral-flow formats for the rapid detection of carbaryl were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and carbaryl hapten-OVA conjugate (test line). Anti-carbaryl antibody labeled with colloidal gold particles was firstly incubated with carbaryl. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for flow-through and lateral flow of 50 and l00 μg/L, respectively. The assay time for both tests was less than 5 min, suitable for rapid testing on-site.     

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and α-zearalenol (α-ZOL) (69%) recognition, while cross-reactivities with α-zearalanol, zearalanone, β-zearalenol and β- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 μg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 μg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and α-ZOL).      

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA–BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL⁻¹, and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74–110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL⁻¹ for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R ² = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.     

Correlation between different clean-up methods and analytical techniques performances to detect Ochratoxin A in wine

Three different clean-up methods and two analytical techniques were compared to determine Ochratoxin A (OTA) in wines. The first clean-up used a MycoSep column, the second an immunoaffinity column (IAC) and the third consisted in a liquid-liquid extraction (LLE) using dichloromethane in acid conditions. Meanwhile, two different OTA determination techniques were also evaluated: a HPLC analysis using a fluorescence detector and an enzyme-linked immunosorbent assays (ELISA) method.Correlations between clean-up methods and analytical techniques to determine OTA in wine were made evaluating linearity, accuracy and precision.Both the two first clean-up methods (solid-phase extraction, SPE) showed a good linear fit (r² = about 0.9999), followed by LLE. The use of immunoaffinity columns showed the best recoveries, even if also the SPE with MycoSep showed good recoveries while the LLE recoveries were the worst ones. The HPLC analysis showed good precision and accuracy, while ELISA method, even with a sufficient linearity, generally underestimated OTA content in wines.     

Exposure assessment to ochratoxin A from the consumption of Italian and Hungarian wines

A total of 267 wine samples including 19 dessert, 186 red, 11 rosé and 51 white produced mostly in the years 1997–2002 in Italian and Hungarian regions were analyzed for ochratoxin A (OTA) using inmunoaffinity column (IAC) clean-up and HPLC with fluorimetric detection. None of Hungarian wine samples were contaminated with this mycotoxin. For Italian red wines, 84% of the samples were positive for OTA ranged from 0.01 to 4.00 ng/mL. Furthermore, OTA was detected in 63% of dessert, in 56% of rosé and in 19% of white wine samples ranged from 0.01 to 1.64, from 0.01 to 1.04 and from 0.01 to 0.21 ng/mL, respectively. A study of OTA daily exposure assessment in Italian wines was also carried out outlining a quite low contribution to the overall daily intake.     

Validation of two analytical methods for the determination of ochratoxin A by reversed-phased high-performance liquid chromatography coupled to fluorescence detection in musts and sweet wines from Andalusia

Some Spanish sweet wines are made from raisins, grapes dried by direct exposure to the sun after picking. This drying process can encourage ochratoxin A (OTA) formation. OTA is a mycotoxin formed by several fungi. It has been linked to nephropathy in humans, and may have a long half-life in humans. The aim of this study is to develop and to apply two procedures for the analysis of OTA in grape musts (during the raisining process) and sweet wines, respectively. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to fluorescence detection (FLD) was employed in both analytical methods. In grape must, the method involves the direct injection of the sample in a HPLC-FLD system without any kind of prior clean-up procedure. The complexity of the sweet wine samples requires a solid-phase extraction (SPE) clean-up on a C18 column which enables the OTA to be isolated from the matrix. The methods used were statistically validated. The validation also included the comparison of the slopes of the curve obtained with standards and the regression curves obtained by the addition of a standard. Two different studies of standard additions were conducted. One method was validated without sample preparation and it was applied to must samples. The other method was validated with SPE extraction and it was applied to sweet wine samples. Recovery was always better than 89.69%. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were established at 0.22 and 0.77 μg l⁻¹, respectively. In general, the analytical data obtained provided good results at the sub-μg l⁻¹ concentration level.     

Novel gel-based rapid test for non-instrumental detection of ochratoxin A in beer

A rapid easy-to-use immunoassay was optimised for the non-instrumental detection of ochratoxin A (OTA) in beer. The analytical method involves preconcentration on the immunoaffinity layer inside a column followed by direct competitive ELISA detection in the same layer. The visual cut-off value, i.e. the lowest OTA concentration resulting in no colour development, was 0.2 μg L^-1. Assay validation was performed using samples spiked with OTA. Thirty-seven naturally contaminated samples were screened with the gel-based method developed and no false-negative results were obtained. The method described offers a simple, rapid and cost-effective screening tool, thus contributing to better health protection of consumers. Figure Gel-based immunoassay of spiked beer samples.     

Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay

Rapid-response analytical tests that can be performed at the point of sampling are based on a visual detection system. The influence of different factors on the signal generation in a membrane-based enzyme immunoassay was investigated. The research was applied to a flow-through immunoassay for the detection of ochratoxin A (OTA). This assay format is a very convenient, simple and fast qualitative screening tool. Conjugates of OTA with horseradish peroxidase (HRP) and alkaline phosphatase (AP) were used as enzyme tracers. A new conjugate OTA-AP has been synthesized in our laboratory and its performance in the assay was compared with that of OTA-HRP. Different substrate systems for HRP and AP were compared. Several reagents, including polymers and surfactants, were tested for their possible effect on signal generation with the use of OTA-HRP conjugate. Polymers such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) 6000 exerted a favourable effect on signal amplification, whereas surfactants negatively affected assay performance. The highest signal amplification (30–70% compared to the standard assay procedure) was achieved using 0.5% PVA in tetramethylbenzidine (TMB) Colorburst substrate solution and phosphate-buffered saline (PBS) for the washing step. It allowed more reliable visual estimation of the results from OTA-HRP assay. Exclusion of the detergent (Tween 20) from the washing solution exerted a favourable effect on assay performance using both enzyme tracers. The assay using OTA-HRP was more susceptible to matrix interferences than the assay with OTA-AP. Signal development in the matrix was better for the OTA-AP assay and visual estimation of the results was easier to perform in this case. For the analysis of spiked wheat samples, OTA-AP conjugate gave a more sensitive, stable and reproducible assay with a cut-off level of 4 μg kg⁻¹ for OTA. The application of the new OTA-AP conjugate resulted in improved assay performance for the food samples.     

A gel-based visual immunoassay for non-instrumental detection of chloramphenicol in food samples

A gel-based non-instrumental immuno-affinity assay was developed for the rapid screening of chloramphenicol (CAP) in food samples with the limit of detection (LOD) of 1 μg L⁻¹. The immuno-affinity test column (IATC) consisted of a test layer containing anti-CAP antibody coupled gel, and a control layer with anti-HRP antibody coupled gel. Based on the direct competitive immuno-reaction and the horseradish peroxidase enzymatic reaction, the test results could be evaluated visually. Basically, blue color development represented the negative results, while the absence of color development represented the positive results. In this study, CAP spiked samples of raw milk, pasteurized milk, UHT milk, skimmed milk powder, acacia honey, date honey, fish and shrimp were tested. Little or none sample pretreatment was required for this assay. The whole procedure was completed within 10 min. In conclusion, the gel-based immuno-affinity test is a simple, rapid, and promising on-site screening method for CAP residues in food samples, with no instrumental requirement.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography

Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium verrucosum. It has been found in foods and feed all over the world. There is a great concern about OTA because it is nephrotoxic and probably, carcinogenic to humans. Most of analytical methods developed for OTA in wine, beer and other products are based on LC with fluorescence detection (LC-FLD). In the present work, various procedures for extraction and/or clean-up for determination of OTA in musts, wine and beer by LC-FLD were compared: (1) dilution with polyethylen glycol 8000 and NaHCO3 solution and clean-up an on immunoaffinity column (IAC); (2) extraction with chloroform and IAC clean-up; solid-phase extraction (SPE) on (3) reversed-phase (RP) C18; (4) RP phenylsilane and (5) Oasis HLB cartridges. SPE on phenylsilane and Oasis HLB have not been reported for OTA analysis in beverages. The same LC-FLD conditions and concentration ratio were used. The former procedure was simple, rapid and provided flat baselines, free from most impurity peaks, high OTA recoveries and quite repeatable results. RP C18 using methanol-acetic acid (99.5:0.5) as elution solvent provided good recoveries and precision, thus becoming a cheaper but interesting alternative at 0.1-1 ng/ml spiking levels. Oasis HLB cartridges were usually better than phenylsilane. Possible binding of OTA to proteins or other components was tested by acid treatment before extraction but no significant differences with controls appeared.     

Concentration of ochratoxin A in wines from supermarkets and stores of Valencian Community (Spain)

Ochratoxin A (OTA) is a mycotoxin produced by fungi species belonging to the genera Aspergillus and Penicillium being isolated in alcoholic beverages. The aim of this work is developed and applied a procedure for the analysis of OTA in wines. An analytical method based on immunoaffinity column (IAC) for clean-up, liquid chromatography with fluorescence detection (LC-FD), and LC-FD after of OTA methylation was used to determine the occurrence of OTA in wines. Recoveries of this mycotoxin spiked to red wines at 0.5 ng/ml level were >90% with an average of relative standards deviations of 4%. Furthermore, 116 wine samples from designation of origin (DO) and three samples from food stores of Valencian Community (Spain) were examined for the occurrence of OTA being the levels of this mycotoxin ranged from <0.01 to 0.76 ng/ml. Finally, the estimated daily intake of OTA in this study was 0.15 ng/kg bw per day.     

Determination of ochratoxin A in wine and beer by immunoaffinity column cleanup and liquid chromatographic analysis with fluorometric detection: collaborative study.

The accuracy, repeatability, and reproducibility characteristics of a liquid chromatographic method for the determination of ochratoxin A (OTA) in white wine, red wine, and beer were established in a collaborative study involving 18 laboratories in 10 countries. Blind duplicates of blank, spiked, and naturally contaminated materials at levels ranging from < or =0.01 to 3.00 ng/mL were analyzed. Wine and beer samples were diluted with a solution containing polyethylene glycol and sodium hydrogen carbonate, and the diluted samples were filtered and cleaned up on an immunoaffinity column. OTA was eluted with methanol and quantified by reversed-phase liquid chromatography with fluorometric detection. Average recoveries from white wine, red wine, and beer ranged from 88.2 to 105.4% (at spiking levels ranging from 0.1 to 2.0 ng/mL), from 84.3 to 93.1% (at spiking levels ranging from 0.2 to 3.0 ng/mL), and from 87.0 to 95.0% (at spiking levels ranging from 0.2 to 1.5 ng/mL), respectively. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 6.6 to 10.8% for white wine, from 6.5 to 10.8% for red wine, and from 4.7 to 16.5% for beer. Relative standard deviations for between-laboratories reproducibility (RSDR) ranged from 13.1 to 15.9% for white wine, from 11.9 to 13.6% for red wine, and from 15.2 to 26.1% for beer. HORRAT values were < or =0.4 for the 3 matrixes.     

Ochratoxin A survey in Portuguese wine by LC-FD with direct injection

Wine and grape juices were identified as one of the most important sources of ochratoxin A (OTA), a mycotoxin with diverse toxic effects that naturally appears in food and foodstuffs all over the world.The aim of this study was to assess the OTA levels in Portuguese wines through the application of a simple and accurate method based on liquid chromatography (LC) with direct injection, followed by fluorescence detection (FD).Randomly selected wine samples were used to evaluate the performance of direct injection as efficient, fast, inexpensive and safe sample preparation method. The proposed method was successfully validated. The limit of quantification (LOQ) was 1.0 μg/L and OTA recoveries from wine samples, spiked at the three fortification levels, were higher than 85.4%, with RSDs lower than 9.6% for both red and white wines. The presence of OTA was confirmed by methyl ester derivatization followed by LC analysis.Data on OTA levels were obtained for 60 Portuguese red and white wine samples. OTA was found in 12 samples, nine (26%) red wine samples and three (12%) white wine samples. Only one red wine sample and one white wine sample presented a contamination level above the LOQ, with 1.23 and 2.4 μg/L, respectively. It should be pointed out that this white wine sample exceeded the EC maximum permitted level of 2.0 μg/L. The safe dose established as 120 ng/kg body weight/week was not exceeded by the weekly intake estimated for the samples contaminated above the LOQ.     

Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Comparison of different immunoaffinity clean-up procedures for high-performance liquid chromatographic analysis of ochratoxin A in wines

Three immunoaffinity clean-up procedures to analyse ochratoxin A (OTA) in wines were compared. The direct wine clean-up with Ochraprep and OchraTest columns gave equivalent results in terms of recovery and precision if compared with the reference procedure involving a preliminary extraction of OTA with chloroform. OTA quantification limit in wine ranged from 0.020 to 0.045 microg/l. The 'on-flow' OTA emission spectrum (excitation 333 nm) showed a maximum at 460 nm and could be used to confirm the quantitative results. The analysis of 11 red and white wines gave no significant quantitative differences between the three clean-up techniques.     

Modified magnetic nanoparticles in an electrochemical method for the ochratoxin A determination in Vitis vinifera red grapes tissues

This work described the development and characterization of an electrochemical method using square wave voltammetry (SWV) combined with the use of modified magnetic nanoparticles (MNPs), which had shown a rapid and sensitive determination of ochratoxin A (OTA) in wine grapes (Cabernet Sauvignon, Malbec and Syrah) post-harvest tissues. The wine grapes were inoculated with Aspergillus ochraceus to obtain OTA in artificially infected samples. The OTA was directly determined using square-wave voltammetry. The current obtained is directly proportional to the concentration of OTA present in the samples. This method has been used for OTA determination in wine grapes and it was validated against a commercial ELISA test kit. The limits of detection calculated for electrochemical detection and the ELISA were 0.02 and 1.9 μg kg⁻¹, respectively and the coefficients of variation for accuracy and precision dates were below 5.5%. This method promises to be suitable for the detection and quantification of OTA in apparently healthy fruits post-harvest for assuring safety and quality of food as well as consumer's health.