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Development of an immunochromatographic strip test for the rapid simultaneous detection of deoxynivalenol and zearalenone in wheat and maize
Food Control 28 (2012) 7e12
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Development of an immunochromatographic strip test for the rapid simultaneous detection of deoxynivalenol and zearalenone in wheat and maize Zhi-Bing Huang a, Yang Xu a, *, Lai-Sheng Li b, Yan-Ping Li a, Hong Zhang a, Qing-Hua He a a b
State Key Laboratory of Food Science and Technology, Sino-Germany Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China Center of Analysis and Testing, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2011 Received in revised form 5 April 2012 Accepted 28 April 2012
A colloidal gold immunochromatographic strip (ICS) test based on competitive format was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN) in wheat and maize samples. For gold-based ICS test, antigen (DON-CBSA and ZEN-BSA) and goat anti-mouse IgG were respectively drawn on NC membrane as two test line (T1 and T2 line) and control line (C line), respectively. Monoclonal antibody (MAb)-gold conjugates (anti-DON MAb-gold and anti-ZEN MAb-gold) were sprayed onto the conjugate pad. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with 50% methanolewater (v/v) by shaking for 3 min and the extract directly used without further cleanup steps. Matrix interference was eliminated by 2.5-fold dilutions of water extracts with buffer. The ICS test, which has cut-off levels of 100 ng/mL (1000 mg/kg) and 6 ng/mL (60 mg/kg) for DON and ZEN, respectively, and can be completed in 5 min. Analysis of DON and ZEN in wheat and maize samples revealed that data obtained from ICS test were in a good agreement with those obtained from ELISA and instrumental analysis (GC/MS for DON determination and HPLC for ZEN determination). The results demonstrate that the ICS test can be used as a reliable, rapid, cost-effective and convenient qualitative tool for on-site screening technique for the simultaneous determination of DON and ZEN in wheat and maize samples without special instrumentation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Colloidal gold Immunochromatographic strip Simultaneous detection Deoxynivalenol Zearalenone Wheat Maize
1. Introduction Mycotoxins are toxic metabolites produced under particular environmental conditions by fungi either in the field or storage of agricultural commodities (Shephard, 2008). Among the most prominent mycotoxins are Alternaria toxins, aflatoxins, ergot alkaloids, fumonisins, ochratoxins, resorcyclic acid lactones, and trichothecenes (Schenzel, Schwarzenbach, & Bucheli, 2010). Deoxynivalenol (DON) and zearalenone (ZEN) are secondary metabolites produced largely by several species of Fusarium fungi, which are widespread in nature and commonly contaminate many cereal grains such as wheat, maize, corn, barley, and other cereal grains (Cetin & Bullerman, 2006). The simultaneous occurrence of DON with other Fusarium toxins, mainly type B trichothecenes and zearalenone, has been reported for various commodities (Kolosova, De Saeger, Sibanda, Verheijen, & Van Peteghem, 2007). DON, for instance, which is a potent protein synthesis inhibitor, is postulated to act as an inhibitor of plant defense response genes (Beyer,
* Corresponding author. Tel.: þ86 (0) 791 8329479; fax: þ86 (0) 791 8333708. E-mail addresses: [email protected], [email protected] (Y. Xu). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.04.035
Verreet, & Ragab, 2005; Neuhof, Koch, Rasenko, & Nehls, 2008; Rotter, Prelusky, & Pestka, 1996). Several adverse effects of DON include dose-dependent induction of feed refusal, diarrhea and emesis in livestock, especially in swine (Kolosova et al., 2007). ZEN is a nonsteroidal estrogenic mycotoxin and has been associated with early puberty, hyperplastic and neoplastic endometrium, and human cervical cancer (Shim, Dzantiev, Eremin, & Chung, 2009). In addition, several studies have been conducted and have shown that ZEN is cytotoxic and exhibits a genotoxic potential in vitro and in vivo through induction of micronuclei, chromosome aberrations, DNA fragmentation, cell cycle arrest, etc (Abbes et al., 2007; Abid-Essefi et al., 2003; Boussema-Ayed, Ouanes, Bacha, & Abid, 2007; Ouanes et al., 2003). As a result, most countries have set a legal limit for DON in cereals and many others for cereal products. For example, in the United States and China a maximum limit for the total amount of DON in cereal products for human consumption is set 1000 mg/kg, and the U.S. Food and Drug Administration has no legal regulations for zearalenone, but in China legal limits for zearalenone is set 60 mg/kg (Meneely et al., 2010; Shim, Kim, & Chung, 2009). To detection related mycotoxins, several analytical methods including HPLC, LC/ MS, GC, GC/MS, and immunoassay have been developed over the
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past decade (Kolosova et al., 2008). Chromatographic techniques are sensitive and are specific tools to simultaneously detect mycotoxins (Desmarchelier et al., 2010; Ediage, Di Mavungu, Monbaliu, Van Peteghem, & De Saeger, 2011; Monbaliu et al., 2010; Romero-Gonzalez, Martinez Vidal, Aguilera-Luiz, & Frenich, 2009; Schenzel et al., 2010), however, chromatographic methods are unsuitable for routine screening of large sample numbers, because they suffer from being time-consuming, laborious, and multicomplex. Most immunoassay has often been limited to laboratories equipped with tools and devices, and they are unsuitable for on-site detection. Therefore, the development of a simple, rapid and low cost analytical methods for these mycotoxins was urgently in need. In the past few years, ICS test has increasingly gained interest in the field of food safety due to the potential for fast and simple on-site application. The ICS test offers a simple and rapid analysis and combines several benefits, such as a user-friendly format, long-term stability over a wide range of climates, and cost-effectiveness (Kolosova et al., 2007; Shim, Dzantiev, et al., 2009; Shim, Kim, et al., 2009). These characteristics make it ideally suited for onsite screening by untrained personnel. Immunochromatographic strips have been used for the detection of small molecular toxins such as ochratoxin A (Cho et al., 2005; Liu, Tsao, Wang, & Yu, 2008), fumonisin B1 (Wang, Quan, Lee, & Kennedy, 2006), aflatoxin B1 (Delmulle, De Saeger, Sibanda, Barna-Vetro, & Van Peteghem, 2005) and T-2 toxin (Molinelli et al., 2008). However, most of these studies are limited only for the detection of a single analyte in sample. One of the latest trends in analytical chemistry is the simultaneous detection of multiple analytes (Shim, Dzantiev, et al., 2009; Shim, Kim, et al., 2009). The merit of simultaneous detection methods is that they can reduce the time and cost per analysis, allow for simpler assay protocols, and decrease the sample volume required (Kolosova et al., 2007). Recently, a few immunochromatographic assay for the rapid simultaneous detection of mycotoxins has been reported (Kolosova et al., 2007; Saha, Acharya, Roy, Shrestha, & Dhar, 2007; Shim, Dzantiev, et al., 2009; Shim, Kim, et al., 2009). So far, there is only one literature about simultaneous detection of DON and ZEN. In this literature, the assay procedure could be accomplished within 10 min, and cut-off levels of 1500 mg/kg and 100 mg/kg for DON and ZEN, respectively, were observed (Kolosova et al., 2007). However, the cut-off levels of method are unsuitable for simultaneous detection of DON and ZEN in cereals for China legal limits for DON (1000 mg/kg) and ZEN (60 mg/kg). In the current study, we improved the preparation of an ICS test for rapid simultaneous detection of DON and ZEN in wheat and maize samples. As confirmed by the results from ELISA and instrumental analysis, ICS test has been demonstrated to be simple and rapid simultaneous screening method for the detection of DON and ZEN on farm or at feed mills. 2. Materials and methods 2.1. Materials and chemicals DON, ZEN, bovine serum albumin (BSA), Tween-20, sucrose, mycose, polyvinylpyrrolidone (PVP), sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), polyethylene glycol (PEG MW ¼ 1500 and 20,000) were purchased from Sigma Chemical Co. (St. Louis, MO). Goat anti-mouse IgG was obtained from SinoAmerican Biotechnology Co. (Luoyang, China). The anti-DON MAb and anti-ZEN MAb were prepared according to methods previously described (Liu et al., 2008). DON-CBSA and ZEN-BSA conjugate as well as the monoclonal antibodies against DON (anti-DON MAb) and ZEN (anti-ZEN MAb) were prepared in our laboratory (Xu et al., 2010). The anti-DON MAb and anti-ZEN MAb were purified using
Protein-G Sepharose Fast Flow Columns (Amersham, NJ, USA). All other chemicals and solvents were of analytical grade or better and were obtained from Beijing Chemical Reagent Co. (Beijing, China). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Stock solutions were prepared by dissolving 1.0 mg of DON in 1.0 mL of deionized water and 1.0 mg of ZEN in 1.0 mL of methanol, and then kept at 20 C for further dilution. The NC membrane Prima 40 (P40) was obtained from Whatman (Middlesex, UK). The colloidal gold (40 nm in diameter), the sample pad, the conjugation pad and the absorbent pad were obtained from Jiening Bio. Co. (Shanghai, China). Immunoaffinity columns for ZEN (ZearalaTest) were supplied from Vicam (Watertown, MA, USA). 2.2. Apparatus The equipment used for spraying and cutting strip tests was purchased from BioDot (Irvine, CA, USA). The BioDot system consisted of two BioJets Quanti 3000 and one Airjet Quanti 3000 attached on a BioDot XYZ-3000 (Irvine, CA, USA) dispensing platform. The guillotine cutter (model CM 4000) was supplied by BioDot (Irvine, CA, USA). Maxisorp polystyrene 96-well plates were purchased from Nunc (Roskilde, Denmark). Immunoassay absorbance was read with a Multiskan MK3 Spectrum purchased from Thermo. A Sigma 2K15 centrifuge from SigmaeAldrich (St. Louis, MO, USA) was used for centrifugation of colloidal gold-MAb conjugates. UVevisible spectra were obtained by using an Ultrospec 4300 Pro UV/Visible Spectrophotometer (Amersham, NJ, USA). For DON determination, the GC/MS method was carried out as previously described (Xu et al., 2010). For ZEN determination, the HPLC system consisted of a Waters 510 solvent delivery pump (Waters, Milford, MA, USA), a 7725 manual injector system equipped with a 20 mL loop, a model 2475 multiwavelength fluorescence detector (Waters, Milford, MA, USA). Fluorescence detection was performed with a 2475 multi-wavelength fluorescence detector with lex ¼ 274 nm and lem ¼ 440 nm. Chromatographic separation was achieved at room temperature using a 250 4.6 mm i.d., 5 mm Symmetry C18 column (Waters, Milford, MA, USA), with isocratic elution of acetonitrileeH2O (70:30, v/v) at a flow rate of 0.80 mL/min. Aliquots of 20 mL standard or samples solutions were injected into the HPLC for the determination. All injections were repeated at least three times. 2.3. Preparation of colloidal gold-MAb conjugates Anti-DON MAb and anti-ZEN MAb prepared in our laboratory were purified from mouse ascitic fluid using a caprylic acid and ammonium sulfate method (Guo et al., 2010). These MAbs were purified further by affinity chromatography using Protein-G Sepharose Fast Flow Columns (Amersham, NJ, USA). Each antibody was conjugated to colloidal gold (40 nm) as previously described (Xu et al., 2010). Briefly, the pH of the colloidal gold solution for anti-DON MAb or anti-ZEN MAb conjugation was adjusted to pH 7.0 with 0.1 M K2CO3. With gentle stirring, anti-DON MAb (100 mg/mL, 1.0 mL) or anti-ZEN MAb (100 mg/mL, 0.5 mL) was added dropwise to the colloidal gold solution (10 mL). After reacting for 30 min, 1 mL of 10% (w/v) BSA was added to block excess reactivity of the gold colloid, and further reacted for 15 min, and then the reaction mixture was centrifuged at 10,000 rpm (8497g) at 4 C for 25 min. The clear supernatant was carefully removed, and gold pellets were resuspended in 1 mL of conjugate storage buffer (5% sucrose, 0.2% BSA, 0.3% PVP, 1% mycose, and 0.05% sodium azide in 0.01 M PBS). From above the process, the anti-DON MAb-gold and anti-ZEN MAb-gold were obtained, and then stored at 4 C until use.
Z.-B. Huang et al. / Food Control 28 (2012) 7e12
2.4. Preparation of immunochromatographic strip The preparation of ICS procedure was carried out as previously described (Xu et al., 2010). An ICS test consists of the following: sample pad, conjugate pad, NC membrane, absorbent pad and backing card. The sample pads (GF-6) and the conjugate pads were treated with 0.01 M PBS (pH 7.4) and with 0.01 M PBS (pH 7.4) containing 5% sucrose, 0.2% BSA, 0.3% PVP, 1% mycose and 0.05% sodium azide, respectively, and then vacuum-dried at 37 C overnight. A total of 5 mL per 1 cm of the mixture 1:1 (v/v) of two colloidal gold-MAb conjugates (anti-DON MAb-gold and anti-ZEN MAb-gold conjugates) diluted three times with 0.01 M PBS (pH 7.4) containing 5% sucrose, 0.2% BSA, 0.3% PVP, 1% mycose and 0.05% sodium azide, was jetted on the treated conjugate pad by using the BioDot XYZ Platform, and then vacuum-dried at 37 C for 2 h. A total of 0.74 mL per 1 cm of DON-CBSA (1.5 mg/mL) conjugate and ZEN-BSA (0.8 mg/mL) conjugate and goat anti-mouse IgG antibody (1.5 mg/mL) were sprayed onto NC membrane (Prima 40, 25 300 mm) as the two test lines (T1 and T2 line) and control line (C line), respectively, by BioDot XYZ Platform, and then vacuumdried at 37 C for 2 h. The distance between lines was 3e4 mm. The sample pad, conjugate pad, NC membrane, absorbent pad and backing card were assembled as the test strip as following: essentially, the NC membrane was adhered to a backing card. The conjugate pad was then secured to the backing card by overlapping 2 mm with the NC membrane. Similarly, the sample pad was also pasted on the backing card by overlying 2 mm with conjugate pad. The absorbent pad was pasted on the top of the membrane sheet. The whole assembled sheet was cut lengthways with an automatic cutter and divided into strips (5 mm 60 mm). The strips were ready for use in the assay and stored under dry conditions at room temperature until use. 2.5. Principle of immunochromatographic strip test The developed ICS test was based on a competitive immunoassay theory. In the absence of DON and ZEN in the sample solution, anti-DON MAb-gold and anti-ZEN MAb-gold conjugates freely migrate into the NC membrane and form antibodyeantigen complexes with the immobilized DON-CBSA and ZEN-BSA conjugates, respectively, thereby forming two visible red line (T1 and T2) on the test zone. In contrast, if the sample contains sufficient DON (more than 100 ng/mL) and ZEN (more than 6 ng/mL), no red line was observed in the corresponding zone. In any assay, the control zone should always show a red color line (C line) under an accurate operation regardless of the presence of DON and ZEN.
9
milliliter of the filtrate was aspirated, diluted with 3 mL distilled water, and then directly subjected to ELISA kits (RidascreenÒ Fast DON ELISA). For ICS test, 1 mL the resulting filtrate was diluted 2.5fold with 0.07 M PBS containing 0.36% Tween-20 and 0.036% anion surfactant, and then 100 mL of the solution was pipetted onto the sample pad of the strip for analysis. 2.8. Analysis of samples A total of 51 natural samples were extracted and tested by the ICS method. For DON detection, the results were confirmed by ELISA and GC/MS (Xu et al., 2010), and for ZEN detection, the results were confirmed by ELISA and HPLC with immunoaffinity columns clear up (Nuryono, Noviandi, Böhm, & Razzazi-Fazeli, 2005). 3. Results and discussion 3.1. Feasibility of multianalysis of DON and ZEN It was known that the haptens of DON and ZEN show great differences in chemical structure. The different corresponding coating antigens for DON or ZEN could be fixed at different sites as respective test lines on the strip. Fig. 1 shows the specificity of antiDON MAb-gold and anti-ZEN MAb-gold conjugates for corresponding test lines. DON-CBSA and ZEN-BSA conjugates were immobilized at difference positions on the NC membrane as respective test lines. The color development was observed at both test lines and control line, when the mixture of the anti-DON MAbgold and anti-ZEN MAb-gold conjugates was applied to the conjugated pad and assay was run with 0.01 M PBS containing 20% methanol and 0.05% anion surfactant (Fig. 1A). However, when anti-DON MAb-gold conjugate (or anti-ZEN MAb-gold conjugate) was added to the conjugate pad, only the color of the DON test line (or ZEN test line) and control line developed. When the application of the DON/ZEN mixture (150/8 ng/mL) to the ICS test treated with the anti-DON MAb-gold and anti-ZEN MAb-gold conjugates resulted in no color development at the test lines (Fig. 1A). It could be concluded that there was no cross-reaction for multianalysis of DON and ZEN using the ICS test. It should be feasible to detect DON and ZEN by combining different conjugates in one NC membrane.
2.6. Detection of standard solution Several concentrations of DON (0, 50, 100, 150, 200 ng/mL), ZEN (0, 2, 4, 6, 8 ng/mL), and DON/ZEN mixtures (0/0, 25/2, 50/4, 100/6, 200/8 ng/mL) were dissolved with 0.01 M PBS (pH 7.4) containing 0.5% Tween-20 and 0.05% anion surfactant as detergent, and then the detection limit was determined. The application of the strips was put in a 100 mL the solution on the sample pad and allowed to migrate up the membrane. 2.7. Sample pretreatments Samples of wheat and maize were ground in the mechanical mortar for 5 min to pass a 20-mesh screen, then mixed, and 5 g of the ground samples were weighed into a 50 mL polypropylene centrifuge tube, and then mixed with 20 mL of 50% methanolewater (v/v). After severe mixing the suspension for 1e2 min, extracts were filtered through a Whatman 2V filter. One
Fig. 1. Illustration of simultaneous ICS and effect of the anion surfactant on the ICS. C line, control line; T1 line, DON test line; T2 line, ZEN test line. The concentration of anion surfactant in the solution of groups A and B was 0.05% (v/v), and 0, respectively. The concentration of DON/ZEN in the solution (from 1e4) was 0/0, 0/6, 100/0, and 100/ 6 ng/mL, respectively.
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Fig. 2. Detection limit of DON/ZEN with ICS test. A series of dilutions (0, 50, 100, 150, and 200 ng/mL) of DON (A), (0, 2, 4, 6, and 8 ng/mL) ZEN (B), and DON/ZEN mixture solutions (0/0, 25/2, 50/4, 100/6, and 200/8 ng/mL) (C) were made in 0.01 M PBS containing 0.5% Tween-20 and 0.05% anion surfactant. The concentration higher than 100 ng/mL DON and 6 ng/mL ZEN was found to cause a disappearance of red line at the T1 line and T2 line, respectively, and the concentration of DON/ZEN mixture solutions higher than 100/6 ng/mL was found to cause a disappearance of red line at both T1 line and T2 line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2. Development of the ICS test
3.3. Detection limit of DON/ZEN ICS test
The main objective of the ICS test was simultaneous qualitative detection of DON (1 mg/kg) and ZEN (60 mg/kg) contamination at threshold levels, and used in on-site screening of wheat and maize samples. For the development of a sensitive ICS test, the various parameters such as pH of colloidal gold solution for conjugated, the amount of antibody for colloidal gold conjugated, detector reagent, capture reagent and NC membrane were optimized as described in our previous work (Xu et al., 2010). In this study, it was also found that the both anti-DON MAb-gold and anti-ZEN Mab-gold were screened for optimum combination in pH 7.0 to obtain the best sensitivity of the ICS test. It is found that the optimum amount of anti-DON MAb and anti-ZEN MAb was 10 mg and 5 mg for 1 mL colloidal gold, respectively. For capture reagents, the DON-CBSA was applied for DON determination by ICS test according to our previous work (Xu et al., 2010). The two types ZEN-carrier protein conjugates (ZEN-BSA and ZEN-CBSA) on NC membrane were also compared. It was also found the nonspecific bindings were high between ZEN-CBSA and colloidal gold, and the nonspecific reaction was unremoved by adding anion surfactants such as SDS and SDBS to the sample solution. This phenomenon was different from the nonspecific reaction between DON-CBSA and colloidal gold. However, the ZENBSA gave the better sensitivity and signal, and unaffected by anion surfactants such as SDS and SDBS in the sample solution (Fig. 1B). For extraction solvents selection, methanol is often used for the extraction of ZEN from cereals, and water or PBS was used for the extraction of DON from cereals. On the one hand, different methanol concentrations in combination with PBS were investigated for the extraction of DON/ZEN from wheat and maize (data not shown). After 3e5 min extraction procedure with 50% (v/v) methanolePBS for wheat and maize samples, the DON recovery was determined by ELISA and GC/MS and the ZEN recovery was determined by ELISA and HPLC. Results shown that the recovery of DON and ZEN was about 80e86.3% and 81e85.1%, respectively. On the other hand, the methanol tolerances of the ICS test were also investigated with different methanol concentrations (0e40% (v/v) methanolePBS). No different color intensity in test lines was observed from 0 to 20% (v/v) methanolePBS, so we selected 20% (v/v) methanolePBS as a working solution (data not shown). For the samples detection by ICS test, the 50% (v/v) methanolePBS extraction solutions were diluted 2.5-fold with PBS before analysis.
The detection limit is defined as the concentration of DON and ZEN in the solution that causes a complete invisibility of the T1 and T2 line. To confirmation the detection limit of the ICS test, a series of DON and ZEN standard solution were assayed ten times by using the ICS test as described previously. The results were judged by at least three person visualization with 5 min after the reaction started. Fig. 2 shows the sensitivity of the ICS test for different presences of DON and ZEN. When a series of DON standard solutions (0, 50, 100, 150, and 200 ng/mL) were applied to the ICS test, the color of the DON test line disappeared at 100 ng/mL of DON, but the color of the ZEN test line developed in all strips (Fig. 2(A)). When a series of ZEN standard solutions (0, 2, 4, 6, and 8 ng/mL) were tested, the color of the ZEN test line disappeared at 6 ng/mL of ZEN, but the color of the DON test line developed in all strips (Fig. 2(B)). Finally, when a series of DON and ZEN mixture solutions (0/0, 25/2, 50/4, 100/6, and 200/8 ng/mL) were detected, no color development of DON and ZEN test lines was observed at 100 ng/mL and 6 ng/mL of
Fig. 3. Comparison of results of ICS without (A) and with storage for 1 year at 4 C (B) and room temperature (C). The DON/ZEN mixture standards from 1 to 4 were 0/0, 100/ 6, 100/0, and 0/6 ng/mL, respectively.
Z.-B. Huang et al. / Food Control 28 (2012) 7e12
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Table 1 Results of DON and ZEN analysis by GC/MS, HPLC, ELISA, and ICS test in the wheat and maize samples. Maize
GC/MS (mg/kg)
DON kita (mg/kg)
HPLC (mg/kg)
ZEN kita (mg/kg)
ICS test DON/ZEN (n ¼ 3)
Wheat
GC/MS (mg/kg)
DON kita (mg/kg)
HPLC (mg/kg)
ZEN kita (mg/kg)
ICS test DON/ZEN (n ¼ 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
NDd 1.44 0.11 ND 0.13 2.87 0.35 0.18 ND 0.18 ND ND 0.17 0.10 0.34 0.27 ND ND 0.46 0.14 0.41 ND ND 0.56 1.16 1.09 2.57 2.90 2.35 2.09 2.50
ND 1.68
ND 8.6 12.6 23.1 15.8 ND ND ND 22.1 ND ND ND ND 14.7 21.5 23.3 ND ND ND ND ND ND ND 39.3 87.6 ND 141.7 323.0 415.1 402.3 469.3
ND 10.1 16.3 15.2 18.1 ND ND ND 24.2 ND ND ND ND 20.1 28.3 17.5 ND ND ND ND ND ND ND 32.8 100.2 ND 153.2 289.3 400.4 382.2 500.1
b/, /, / þc/, þ/, þ/ /, /, / /, /, / /, /, / þ/, þ/, þ/ /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.53 1.40 2.59 1.36 0.56 2.68 1.10 1.14 1.01 4.59 ND 1.15 2.90 1.80 2.45 2.12 2.10 1.27 1.58 1.01
1.66 1.57 2.63 1.22 0.47 2.89 1.03 1.05 1.11 4.46
38.2 ND 36.2 67.3 ND 31.2 18.3 36.2 19.4 ND ND 33.2 ND ND ND ND 27.2 48.3 ND ND
35.1 ND 45.1 73.1 ND 20.4 23.2 32.1 18.3 ND ND 21.3 ND ND ND ND 36.4 50.2 ND ND
þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/þ, þ/þ, þ/þ /, /, / þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ /, /, / þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ /, /, /
a b c d e
Screening by RidascreenÒ Fast DON or ZEN ELISA kits. Positive result, T1 or T2 line vanished. Negative result, both C line and T line ( T1 or T2) appeared clearly. Not detected (ND). Limit of determination of DON, 0.2 mg/kg.
DON and ZEN, respectively (Fig. 2(C)). So the detection limit of the ICS test for DON and ZEN was 100 and 6 ng/mL, respectively. 3.4. Stability of ICS After treatments of sample pad, conjugate pads and NC membrane as described above, they were laminated into a sheet of
plastic backing orderly and cut into test strip. The assembly completed ICS were kept at 4 C and room temperature for 1 year in plastic bags containing silica gel desiccant. During the storage, the ICS were tested with the DON/ZEN mixture solutions (0/0, 25/2, 100/6 ng/mL) at an interval of 7 days in the former 3 months, and then an interval of 30 days for the later 7 months. As shown in Fig. 3, the ICS stored at 4 C and room temperature for 1 year gave
Fig. 4. Simultaneous detection of DON and ZEN with ICS in 31 maize (A) and 20 wheat (B) samples.
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the same results at the test lines and C line, when the DON/ZEN mixture standards (0/0, 100/6, 100/0, and 0/6 ng/mL) were assayed. Therefore, the ICS developed in this study can be stored for 1 year at room temperature. The result indicated that the ICS may be suitable as a commercial kit. 3.5. Simultaneous detection of DON and ZEN in wheat and maize samples Thirty-one natural maize samples purchased in local markets and 20 wheat samples provided by Hebei Provincial State Grain and Oil Quality Inspection Station (China) were analyzed using the ICS test and then confirmed with ELISA and instrumental analysis (GC/ MS for DON and HPLC for ZEN determination). Results were shown in Table 1. DON positive results including sample 2, 6, 25, 27, 28, 29, 30 and 31 of maize, and sample 1, 2, 3, 4, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, and 19 of wheat containing range from 1.01 mg/kg to 4.59 mg/kg, in ELISA and GC/MS gave a positive result with one or two red line on the ICS test (Fig. 4). ZEN positive results including sample 25, 27, 28, 29, 30 and 31 of maize, and sample 4 of wheat containing rage from 67.3 mg/kg to 500.1 mg/kg. All the contained wheat and maize samples with DON levels lower than 1000 mg/kg, and ZEN levels lower than 60 mg/kg, indicating that they were DON and ZEN negative, respectively, in the ICS test. Results of samples analysis obtained from the ICS were in a good agreement with those obtained form ELISA and instrumental analysis. This results show that the three methods corresponded well, and the ICS test gave neither false-positive nor false-negative results. 4. Conclusions A rapid ICS test was developed to simultaneously detect DON and ZEN in wheat and maize samples. The ICS test can be used as qualitative tools for the rapid simultaneous screening of DON and ZEN contamination within 10 min (including rapid extraction and assay time) on-site. The ICS test had a visual detection limit of 100 ng/mL for DON and 6 ng/mL for ZEN. The sensitivities of the current assay methods were sufficient to detect DON and ZEN at the maximum residue limit of 1000 mg/kg and 60 mg/kg, respectively, proposed for legislation in China and are suitable for use as rapid simultaneous screening tests for DON and ZEN. Acknowledgments This work was financially supported by grants from the National High Technology Research and Development Program of China (863 Program, No. 2007AA10Z427), and the National Natural Science Foundation of China (31160308 and 31171696), and the Research Foundation for Young Scientists of State Key Laboratory of Food Science and Technology, Nanchang University, China (SKLFQN-201102). References Abbes, S., Quanes, Z., Salah-Abbes, J. B., Abdel-Wahhab, M. A., Oueslati, R., & Bacha, H. (2007). Preventive role of aluminosilicate clay against induction of micronuclei and chromosome aberrations in bonemarrow cells of Balb/C mice treated with zearalenone. Mutation Research, 21, 136e144. Abid-Essefi, S., Baudrimont, I., Hassen, W., Ouanes, Z., Mobio, T. A., Anane, R., et al. (2003). DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by vitamin E. Toxicology, 192, 237e248. Beyer, M., Verreet, J. A., & Ragab, S. M. (2005). Effect of relative humidity on germination of ascospores and macroconidia of Gibberella zeae and deoxynivalenol production. International Journal of Food Microbiology, 98, 233e240. Boussema-Ayed, I., Ouanes, Z., Bacha, H., & Abid, S. (2007). Toxicities induced in cultured cells exposed to zearalenone: apoptosis or mutagenesis? Journal of Biochemical and Molecular Toxicology, 21, 136e144.
Cetin, Y., & Bullerman, L. B. (2006). Confirmation of reduced toxicity of deoxynivalenol in extrusion-processed corn grits by the MTT bioassay. Journal of Agricultural Food Chemistry, 54, 1949e1955. Cho, Y. J., Lee, D. H., Kim, D. O., Min, W. K., Bong, K. T., Lee, G. G., et al. (2005). Production of a monoclonal antibody against ochratoxin A and its application to immunochromatographic assay. Journal of Agricultural and Food Chemistry, 53, 8447e8451. Delmulle, B. S., De Saeger, S. M. D. G., Sibanda, L., Barna-Vetro, I., & Van Peteghem, C. H. (2005). Development of an immunoassay based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. Journal of Agricultural and Food Chemistry, 53, 3364e3368. Desmarchelier, A., Oberson, J. M., Tella, P., Gremaud, E., Seefelder, W., & Mottier, P. (2010). Development and comparison of two multiresidue methods for the analysis of 17 mycotoxins in cereals by liquid chromatography electrospray ionization tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 58, 7510e7519. Ediage, E. N., Di Mavungu, J. D., Monbaliu, S., Van Peteghem, C., & De Saeger, S. (2011). A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples. Journal of Agricultural and Food Chemistry, 59, 5173e5180. Guo, Y. C., Ngom, B., Le, T., Jin, X., Wang, L. P., Shi, D. S., et al. (2010). Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle. Analytical Chemistry, 82, 7550e7555. Kolosova, A. Y., De Saeger, S., Sibanda, L., Verheijen, R., & Van Peteghem, C. (2007). Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of zearalenone and deoxynivalenol. Analytical and Bioanalytical Chemistry, 389, 2103e2107. Kolosova, A. Y., Sibanda, L., Dumoulin, F., Lewis, J., Duveiller, E., Van Peteghem, C., et al. (2008). Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges. Anayltica Chimica Acta, 616, 235e244. Liu, B. H., Tsao, Z. J., Wang, J. J., & Yu, F. Y. (2008). Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip. Analytical Chemistry, 80, 7029e7035. Meneely, J., Fodey, T., Armstrong, L., Sulyok, M., Krska, R., & Elliott, C. (2010). Rapid surface plasmon resonance immunoassay for the determination of deoxynivalenol in wheat, wheat products, and maize-based baby food. Journal of Agricultural and Food Chemistry, 58, 8936e8941. Molinelli, A., Grossalber, K., Führer, M., Baumgartner, S., Sulyok, M., & Krska, R. (2008). Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat. Journal of Agricultural and Food Chemistry, 56, 2589e2594. Monbaliu, S., Van Poucke, C., Detavernier, C., Dumoulin, F., Van De Velde, M., Schoeters, E., et al. (2010). Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. Journal of Agricultural and Food Chemistry, 58, 66e71. Neuhof, T., Koch, M., Rasenko, T., & Nehls, I. (2008). Distribution of trichothecenes, zearalenone, and ergosterol in a fractionated wheat harvest lot. Journal of Agricultural and Food Chemistry, 56, 7566e7571. Nuryono, N., Noviandi, C. T., Böhm, J., & Razzazi-Fazeli, E. (2005). A limited survey of zearalenone in Indonesian maize-based food and feed by ELISA and high performance liquid chromatography. Food Control, 16, 65e71. Ouanes, Z., Abid, S., Ayed, I., Anane, R., Mobio, T., Creppy, E. E., et al. (2003). Induction of micronuclei by zearalenone in vero monkey kidney cells and in bone marrow cells of mice: protective effect of vitamin E. Mutation Research, 538, 63e70. Romero-Gonzalez, R., Martinez Vidal, J. L., Aguilera-Luiz, M. M., & Frenich, A. G. (2009). Application of conventional solid-phase extraction for multimycotoxin analysis in beers by ultrahigh-performance liquid chromatography-tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 57, 9385e9392. Rotter, B. A., Prelusky, D. B., & Pestka, J. J. (1996). Toxicology of deoxynivalenol (vomitoxin). Journal of Toxicology and Environmental Health, 48, 1e34. Saha, D., Acharya, D., Roy, D., Shrestha, D., & Dhar, T. K. (2007). Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples. Anayltica Chimica Acta, 584, 343e349. Schenzel, J., Schwarzenbach, R. P., & Bucheli, T. D. (2010). Multi-residue screening method to quantify mycotoxins in aqueous environmental samples. Journal of Agricultural and Food Chemistry, 58, 11207e11217. Shephard, G. S. (2008). Determination of mycotoxins in human food. Chemical Society Reviews, 37, 2468e2477. Shim, W. B., Dzantiev, B. B., Eremin, S. A., & Chung, D. H. (2009). One-step simultaneous immunochromatographic strip test for multianalysis of ochratoxin A and zearalenone. Journal of Microbiology and Biotechnology, 19, 83e92. Shim, W. B., Kim, K. Y., & Chung, D. H. (2009). Development and validation of a gold nanoparticle immunochromatographic assay (ICG) for the detection of zearalenone. Journal of Agricultural and Food Chemistry, 57, 4035e4041. Wang, S., Quan, Y., Lee, N., & Kennedy, I. R. (2006). Rapid determination of fumonisin B1 in food samples by enzyme-linked immunosorbent assay and colloidal gold immunoassay. Journal of Agricultural and Food Chemistry, 54, 2491e2495. Xu, Y., Huang, Z. B., He, Q. H., Deng, S. Z., Li, L. S., & Li, Y. P. (2010). Development of an immunochromatographic strip test for the rapid detection of deoxynivalenol in wheat and maize. Food Chemistry, 119, 834e839.
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Development of an immunochromatographic strip test for the rapid simultaneous detection of deoxynivalenol and zearalenone in wheat and maize Zhi-Bing Huang a, Yang Xu a, *, Lai-Sheng Li b, Yan-Ping Li a, Hong Zhang a, Qing-Hua He a a b
State Key Laboratory of Food Science and Technology, Sino-Germany Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China Center of Analysis and Testing, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2011 Received in revised form 5 April 2012 Accepted 28 April 2012
A colloidal gold immunochromatographic strip (ICS) test based on competitive format was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN) in wheat and maize samples. For gold-based ICS test, antigen (DON-CBSA and ZEN-BSA) and goat anti-mouse IgG were respectively drawn on NC membrane as two test line (T1 and T2 line) and control line (C line), respectively. Monoclonal antibody (MAb)-gold conjugates (anti-DON MAb-gold and anti-ZEN MAb-gold) were sprayed onto the conjugate pad. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with 50% methanolewater (v/v) by shaking for 3 min and the extract directly used without further cleanup steps. Matrix interference was eliminated by 2.5-fold dilutions of water extracts with buffer. The ICS test, which has cut-off levels of 100 ng/mL (1000 mg/kg) and 6 ng/mL (60 mg/kg) for DON and ZEN, respectively, and can be completed in 5 min. Analysis of DON and ZEN in wheat and maize samples revealed that data obtained from ICS test were in a good agreement with those obtained from ELISA and instrumental analysis (GC/MS for DON determination and HPLC for ZEN determination). The results demonstrate that the ICS test can be used as a reliable, rapid, cost-effective and convenient qualitative tool for on-site screening technique for the simultaneous determination of DON and ZEN in wheat and maize samples without special instrumentation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Colloidal gold Immunochromatographic strip Simultaneous detection Deoxynivalenol Zearalenone Wheat Maize
1. Introduction Mycotoxins are toxic metabolites produced under particular environmental conditions by fungi either in the field or storage of agricultural commodities (Shephard, 2008). Among the most prominent mycotoxins are Alternaria toxins, aflatoxins, ergot alkaloids, fumonisins, ochratoxins, resorcyclic acid lactones, and trichothecenes (Schenzel, Schwarzenbach, & Bucheli, 2010). Deoxynivalenol (DON) and zearalenone (ZEN) are secondary metabolites produced largely by several species of Fusarium fungi, which are widespread in nature and commonly contaminate many cereal grains such as wheat, maize, corn, barley, and other cereal grains (Cetin & Bullerman, 2006). The simultaneous occurrence of DON with other Fusarium toxins, mainly type B trichothecenes and zearalenone, has been reported for various commodities (Kolosova, De Saeger, Sibanda, Verheijen, & Van Peteghem, 2007). DON, for instance, which is a potent protein synthesis inhibitor, is postulated to act as an inhibitor of plant defense response genes (Beyer,
* Corresponding author. Tel.: þ86 (0) 791 8329479; fax: þ86 (0) 791 8333708. E-mail addresses: [email protected], [email protected] (Y. Xu). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.04.035
Verreet, & Ragab, 2005; Neuhof, Koch, Rasenko, & Nehls, 2008; Rotter, Prelusky, & Pestka, 1996). Several adverse effects of DON include dose-dependent induction of feed refusal, diarrhea and emesis in livestock, especially in swine (Kolosova et al., 2007). ZEN is a nonsteroidal estrogenic mycotoxin and has been associated with early puberty, hyperplastic and neoplastic endometrium, and human cervical cancer (Shim, Dzantiev, Eremin, & Chung, 2009). In addition, several studies have been conducted and have shown that ZEN is cytotoxic and exhibits a genotoxic potential in vitro and in vivo through induction of micronuclei, chromosome aberrations, DNA fragmentation, cell cycle arrest, etc (Abbes et al., 2007; Abid-Essefi et al., 2003; Boussema-Ayed, Ouanes, Bacha, & Abid, 2007; Ouanes et al., 2003). As a result, most countries have set a legal limit for DON in cereals and many others for cereal products. For example, in the United States and China a maximum limit for the total amount of DON in cereal products for human consumption is set 1000 mg/kg, and the U.S. Food and Drug Administration has no legal regulations for zearalenone, but in China legal limits for zearalenone is set 60 mg/kg (Meneely et al., 2010; Shim, Kim, & Chung, 2009). To detection related mycotoxins, several analytical methods including HPLC, LC/ MS, GC, GC/MS, and immunoassay have been developed over the
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past decade (Kolosova et al., 2008). Chromatographic techniques are sensitive and are specific tools to simultaneously detect mycotoxins (Desmarchelier et al., 2010; Ediage, Di Mavungu, Monbaliu, Van Peteghem, & De Saeger, 2011; Monbaliu et al., 2010; Romero-Gonzalez, Martinez Vidal, Aguilera-Luiz, & Frenich, 2009; Schenzel et al., 2010), however, chromatographic methods are unsuitable for routine screening of large sample numbers, because they suffer from being time-consuming, laborious, and multicomplex. Most immunoassay has often been limited to laboratories equipped with tools and devices, and they are unsuitable for on-site detection. Therefore, the development of a simple, rapid and low cost analytical methods for these mycotoxins was urgently in need. In the past few years, ICS test has increasingly gained interest in the field of food safety due to the potential for fast and simple on-site application. The ICS test offers a simple and rapid analysis and combines several benefits, such as a user-friendly format, long-term stability over a wide range of climates, and cost-effectiveness (Kolosova et al., 2007; Shim, Dzantiev, et al., 2009; Shim, Kim, et al., 2009). These characteristics make it ideally suited for onsite screening by untrained personnel. Immunochromatographic strips have been used for the detection of small molecular toxins such as ochratoxin A (Cho et al., 2005; Liu, Tsao, Wang, & Yu, 2008), fumonisin B1 (Wang, Quan, Lee, & Kennedy, 2006), aflatoxin B1 (Delmulle, De Saeger, Sibanda, Barna-Vetro, & Van Peteghem, 2005) and T-2 toxin (Molinelli et al., 2008). However, most of these studies are limited only for the detection of a single analyte in sample. One of the latest trends in analytical chemistry is the simultaneous detection of multiple analytes (Shim, Dzantiev, et al., 2009; Shim, Kim, et al., 2009). The merit of simultaneous detection methods is that they can reduce the time and cost per analysis, allow for simpler assay protocols, and decrease the sample volume required (Kolosova et al., 2007). Recently, a few immunochromatographic assay for the rapid simultaneous detection of mycotoxins has been reported (Kolosova et al., 2007; Saha, Acharya, Roy, Shrestha, & Dhar, 2007; Shim, Dzantiev, et al., 2009; Shim, Kim, et al., 2009). So far, there is only one literature about simultaneous detection of DON and ZEN. In this literature, the assay procedure could be accomplished within 10 min, and cut-off levels of 1500 mg/kg and 100 mg/kg for DON and ZEN, respectively, were observed (Kolosova et al., 2007). However, the cut-off levels of method are unsuitable for simultaneous detection of DON and ZEN in cereals for China legal limits for DON (1000 mg/kg) and ZEN (60 mg/kg). In the current study, we improved the preparation of an ICS test for rapid simultaneous detection of DON and ZEN in wheat and maize samples. As confirmed by the results from ELISA and instrumental analysis, ICS test has been demonstrated to be simple and rapid simultaneous screening method for the detection of DON and ZEN on farm or at feed mills. 2. Materials and methods 2.1. Materials and chemicals DON, ZEN, bovine serum albumin (BSA), Tween-20, sucrose, mycose, polyvinylpyrrolidone (PVP), sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), polyethylene glycol (PEG MW ¼ 1500 and 20,000) were purchased from Sigma Chemical Co. (St. Louis, MO). Goat anti-mouse IgG was obtained from SinoAmerican Biotechnology Co. (Luoyang, China). The anti-DON MAb and anti-ZEN MAb were prepared according to methods previously described (Liu et al., 2008). DON-CBSA and ZEN-BSA conjugate as well as the monoclonal antibodies against DON (anti-DON MAb) and ZEN (anti-ZEN MAb) were prepared in our laboratory (Xu et al., 2010). The anti-DON MAb and anti-ZEN MAb were purified using
Protein-G Sepharose Fast Flow Columns (Amersham, NJ, USA). All other chemicals and solvents were of analytical grade or better and were obtained from Beijing Chemical Reagent Co. (Beijing, China). Deionized water was prepared using a Milli-Q water purification system (Millipore, Bedford, MA, USA). Stock solutions were prepared by dissolving 1.0 mg of DON in 1.0 mL of deionized water and 1.0 mg of ZEN in 1.0 mL of methanol, and then kept at 20 C for further dilution. The NC membrane Prima 40 (P40) was obtained from Whatman (Middlesex, UK). The colloidal gold (40 nm in diameter), the sample pad, the conjugation pad and the absorbent pad were obtained from Jiening Bio. Co. (Shanghai, China). Immunoaffinity columns for ZEN (ZearalaTest) were supplied from Vicam (Watertown, MA, USA). 2.2. Apparatus The equipment used for spraying and cutting strip tests was purchased from BioDot (Irvine, CA, USA). The BioDot system consisted of two BioJets Quanti 3000 and one Airjet Quanti 3000 attached on a BioDot XYZ-3000 (Irvine, CA, USA) dispensing platform. The guillotine cutter (model CM 4000) was supplied by BioDot (Irvine, CA, USA). Maxisorp polystyrene 96-well plates were purchased from Nunc (Roskilde, Denmark). Immunoassay absorbance was read with a Multiskan MK3 Spectrum purchased from Thermo. A Sigma 2K15 centrifuge from SigmaeAldrich (St. Louis, MO, USA) was used for centrifugation of colloidal gold-MAb conjugates. UVevisible spectra were obtained by using an Ultrospec 4300 Pro UV/Visible Spectrophotometer (Amersham, NJ, USA). For DON determination, the GC/MS method was carried out as previously described (Xu et al., 2010). For ZEN determination, the HPLC system consisted of a Waters 510 solvent delivery pump (Waters, Milford, MA, USA), a 7725 manual injector system equipped with a 20 mL loop, a model 2475 multiwavelength fluorescence detector (Waters, Milford, MA, USA). Fluorescence detection was performed with a 2475 multi-wavelength fluorescence detector with lex ¼ 274 nm and lem ¼ 440 nm. Chromatographic separation was achieved at room temperature using a 250 4.6 mm i.d., 5 mm Symmetry C18 column (Waters, Milford, MA, USA), with isocratic elution of acetonitrileeH2O (70:30, v/v) at a flow rate of 0.80 mL/min. Aliquots of 20 mL standard or samples solutions were injected into the HPLC for the determination. All injections were repeated at least three times. 2.3. Preparation of colloidal gold-MAb conjugates Anti-DON MAb and anti-ZEN MAb prepared in our laboratory were purified from mouse ascitic fluid using a caprylic acid and ammonium sulfate method (Guo et al., 2010). These MAbs were purified further by affinity chromatography using Protein-G Sepharose Fast Flow Columns (Amersham, NJ, USA). Each antibody was conjugated to colloidal gold (40 nm) as previously described (Xu et al., 2010). Briefly, the pH of the colloidal gold solution for anti-DON MAb or anti-ZEN MAb conjugation was adjusted to pH 7.0 with 0.1 M K2CO3. With gentle stirring, anti-DON MAb (100 mg/mL, 1.0 mL) or anti-ZEN MAb (100 mg/mL, 0.5 mL) was added dropwise to the colloidal gold solution (10 mL). After reacting for 30 min, 1 mL of 10% (w/v) BSA was added to block excess reactivity of the gold colloid, and further reacted for 15 min, and then the reaction mixture was centrifuged at 10,000 rpm (8497g) at 4 C for 25 min. The clear supernatant was carefully removed, and gold pellets were resuspended in 1 mL of conjugate storage buffer (5% sucrose, 0.2% BSA, 0.3% PVP, 1% mycose, and 0.05% sodium azide in 0.01 M PBS). From above the process, the anti-DON MAb-gold and anti-ZEN MAb-gold were obtained, and then stored at 4 C until use.
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2.4. Preparation of immunochromatographic strip The preparation of ICS procedure was carried out as previously described (Xu et al., 2010). An ICS test consists of the following: sample pad, conjugate pad, NC membrane, absorbent pad and backing card. The sample pads (GF-6) and the conjugate pads were treated with 0.01 M PBS (pH 7.4) and with 0.01 M PBS (pH 7.4) containing 5% sucrose, 0.2% BSA, 0.3% PVP, 1% mycose and 0.05% sodium azide, respectively, and then vacuum-dried at 37 C overnight. A total of 5 mL per 1 cm of the mixture 1:1 (v/v) of two colloidal gold-MAb conjugates (anti-DON MAb-gold and anti-ZEN MAb-gold conjugates) diluted three times with 0.01 M PBS (pH 7.4) containing 5% sucrose, 0.2% BSA, 0.3% PVP, 1% mycose and 0.05% sodium azide, was jetted on the treated conjugate pad by using the BioDot XYZ Platform, and then vacuum-dried at 37 C for 2 h. A total of 0.74 mL per 1 cm of DON-CBSA (1.5 mg/mL) conjugate and ZEN-BSA (0.8 mg/mL) conjugate and goat anti-mouse IgG antibody (1.5 mg/mL) were sprayed onto NC membrane (Prima 40, 25 300 mm) as the two test lines (T1 and T2 line) and control line (C line), respectively, by BioDot XYZ Platform, and then vacuumdried at 37 C for 2 h. The distance between lines was 3e4 mm. The sample pad, conjugate pad, NC membrane, absorbent pad and backing card were assembled as the test strip as following: essentially, the NC membrane was adhered to a backing card. The conjugate pad was then secured to the backing card by overlapping 2 mm with the NC membrane. Similarly, the sample pad was also pasted on the backing card by overlying 2 mm with conjugate pad. The absorbent pad was pasted on the top of the membrane sheet. The whole assembled sheet was cut lengthways with an automatic cutter and divided into strips (5 mm 60 mm). The strips were ready for use in the assay and stored under dry conditions at room temperature until use. 2.5. Principle of immunochromatographic strip test The developed ICS test was based on a competitive immunoassay theory. In the absence of DON and ZEN in the sample solution, anti-DON MAb-gold and anti-ZEN MAb-gold conjugates freely migrate into the NC membrane and form antibodyeantigen complexes with the immobilized DON-CBSA and ZEN-BSA conjugates, respectively, thereby forming two visible red line (T1 and T2) on the test zone. In contrast, if the sample contains sufficient DON (more than 100 ng/mL) and ZEN (more than 6 ng/mL), no red line was observed in the corresponding zone. In any assay, the control zone should always show a red color line (C line) under an accurate operation regardless of the presence of DON and ZEN.
9
milliliter of the filtrate was aspirated, diluted with 3 mL distilled water, and then directly subjected to ELISA kits (RidascreenÒ Fast DON ELISA). For ICS test, 1 mL the resulting filtrate was diluted 2.5fold with 0.07 M PBS containing 0.36% Tween-20 and 0.036% anion surfactant, and then 100 mL of the solution was pipetted onto the sample pad of the strip for analysis. 2.8. Analysis of samples A total of 51 natural samples were extracted and tested by the ICS method. For DON detection, the results were confirmed by ELISA and GC/MS (Xu et al., 2010), and for ZEN detection, the results were confirmed by ELISA and HPLC with immunoaffinity columns clear up (Nuryono, Noviandi, Böhm, & Razzazi-Fazeli, 2005). 3. Results and discussion 3.1. Feasibility of multianalysis of DON and ZEN It was known that the haptens of DON and ZEN show great differences in chemical structure. The different corresponding coating antigens for DON or ZEN could be fixed at different sites as respective test lines on the strip. Fig. 1 shows the specificity of antiDON MAb-gold and anti-ZEN MAb-gold conjugates for corresponding test lines. DON-CBSA and ZEN-BSA conjugates were immobilized at difference positions on the NC membrane as respective test lines. The color development was observed at both test lines and control line, when the mixture of the anti-DON MAbgold and anti-ZEN MAb-gold conjugates was applied to the conjugated pad and assay was run with 0.01 M PBS containing 20% methanol and 0.05% anion surfactant (Fig. 1A). However, when anti-DON MAb-gold conjugate (or anti-ZEN MAb-gold conjugate) was added to the conjugate pad, only the color of the DON test line (or ZEN test line) and control line developed. When the application of the DON/ZEN mixture (150/8 ng/mL) to the ICS test treated with the anti-DON MAb-gold and anti-ZEN MAb-gold conjugates resulted in no color development at the test lines (Fig. 1A). It could be concluded that there was no cross-reaction for multianalysis of DON and ZEN using the ICS test. It should be feasible to detect DON and ZEN by combining different conjugates in one NC membrane.
2.6. Detection of standard solution Several concentrations of DON (0, 50, 100, 150, 200 ng/mL), ZEN (0, 2, 4, 6, 8 ng/mL), and DON/ZEN mixtures (0/0, 25/2, 50/4, 100/6, 200/8 ng/mL) were dissolved with 0.01 M PBS (pH 7.4) containing 0.5% Tween-20 and 0.05% anion surfactant as detergent, and then the detection limit was determined. The application of the strips was put in a 100 mL the solution on the sample pad and allowed to migrate up the membrane. 2.7. Sample pretreatments Samples of wheat and maize were ground in the mechanical mortar for 5 min to pass a 20-mesh screen, then mixed, and 5 g of the ground samples were weighed into a 50 mL polypropylene centrifuge tube, and then mixed with 20 mL of 50% methanolewater (v/v). After severe mixing the suspension for 1e2 min, extracts were filtered through a Whatman 2V filter. One
Fig. 1. Illustration of simultaneous ICS and effect of the anion surfactant on the ICS. C line, control line; T1 line, DON test line; T2 line, ZEN test line. The concentration of anion surfactant in the solution of groups A and B was 0.05% (v/v), and 0, respectively. The concentration of DON/ZEN in the solution (from 1e4) was 0/0, 0/6, 100/0, and 100/ 6 ng/mL, respectively.
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Fig. 2. Detection limit of DON/ZEN with ICS test. A series of dilutions (0, 50, 100, 150, and 200 ng/mL) of DON (A), (0, 2, 4, 6, and 8 ng/mL) ZEN (B), and DON/ZEN mixture solutions (0/0, 25/2, 50/4, 100/6, and 200/8 ng/mL) (C) were made in 0.01 M PBS containing 0.5% Tween-20 and 0.05% anion surfactant. The concentration higher than 100 ng/mL DON and 6 ng/mL ZEN was found to cause a disappearance of red line at the T1 line and T2 line, respectively, and the concentration of DON/ZEN mixture solutions higher than 100/6 ng/mL was found to cause a disappearance of red line at both T1 line and T2 line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2. Development of the ICS test
3.3. Detection limit of DON/ZEN ICS test
The main objective of the ICS test was simultaneous qualitative detection of DON (1 mg/kg) and ZEN (60 mg/kg) contamination at threshold levels, and used in on-site screening of wheat and maize samples. For the development of a sensitive ICS test, the various parameters such as pH of colloidal gold solution for conjugated, the amount of antibody for colloidal gold conjugated, detector reagent, capture reagent and NC membrane were optimized as described in our previous work (Xu et al., 2010). In this study, it was also found that the both anti-DON MAb-gold and anti-ZEN Mab-gold were screened for optimum combination in pH 7.0 to obtain the best sensitivity of the ICS test. It is found that the optimum amount of anti-DON MAb and anti-ZEN MAb was 10 mg and 5 mg for 1 mL colloidal gold, respectively. For capture reagents, the DON-CBSA was applied for DON determination by ICS test according to our previous work (Xu et al., 2010). The two types ZEN-carrier protein conjugates (ZEN-BSA and ZEN-CBSA) on NC membrane were also compared. It was also found the nonspecific bindings were high between ZEN-CBSA and colloidal gold, and the nonspecific reaction was unremoved by adding anion surfactants such as SDS and SDBS to the sample solution. This phenomenon was different from the nonspecific reaction between DON-CBSA and colloidal gold. However, the ZENBSA gave the better sensitivity and signal, and unaffected by anion surfactants such as SDS and SDBS in the sample solution (Fig. 1B). For extraction solvents selection, methanol is often used for the extraction of ZEN from cereals, and water or PBS was used for the extraction of DON from cereals. On the one hand, different methanol concentrations in combination with PBS were investigated for the extraction of DON/ZEN from wheat and maize (data not shown). After 3e5 min extraction procedure with 50% (v/v) methanolePBS for wheat and maize samples, the DON recovery was determined by ELISA and GC/MS and the ZEN recovery was determined by ELISA and HPLC. Results shown that the recovery of DON and ZEN was about 80e86.3% and 81e85.1%, respectively. On the other hand, the methanol tolerances of the ICS test were also investigated with different methanol concentrations (0e40% (v/v) methanolePBS). No different color intensity in test lines was observed from 0 to 20% (v/v) methanolePBS, so we selected 20% (v/v) methanolePBS as a working solution (data not shown). For the samples detection by ICS test, the 50% (v/v) methanolePBS extraction solutions were diluted 2.5-fold with PBS before analysis.
The detection limit is defined as the concentration of DON and ZEN in the solution that causes a complete invisibility of the T1 and T2 line. To confirmation the detection limit of the ICS test, a series of DON and ZEN standard solution were assayed ten times by using the ICS test as described previously. The results were judged by at least three person visualization with 5 min after the reaction started. Fig. 2 shows the sensitivity of the ICS test for different presences of DON and ZEN. When a series of DON standard solutions (0, 50, 100, 150, and 200 ng/mL) were applied to the ICS test, the color of the DON test line disappeared at 100 ng/mL of DON, but the color of the ZEN test line developed in all strips (Fig. 2(A)). When a series of ZEN standard solutions (0, 2, 4, 6, and 8 ng/mL) were tested, the color of the ZEN test line disappeared at 6 ng/mL of ZEN, but the color of the DON test line developed in all strips (Fig. 2(B)). Finally, when a series of DON and ZEN mixture solutions (0/0, 25/2, 50/4, 100/6, and 200/8 ng/mL) were detected, no color development of DON and ZEN test lines was observed at 100 ng/mL and 6 ng/mL of
Fig. 3. Comparison of results of ICS without (A) and with storage for 1 year at 4 C (B) and room temperature (C). The DON/ZEN mixture standards from 1 to 4 were 0/0, 100/ 6, 100/0, and 0/6 ng/mL, respectively.
Z.-B. Huang et al. / Food Control 28 (2012) 7e12
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Table 1 Results of DON and ZEN analysis by GC/MS, HPLC, ELISA, and ICS test in the wheat and maize samples. Maize
GC/MS (mg/kg)
DON kita (mg/kg)
HPLC (mg/kg)
ZEN kita (mg/kg)
ICS test DON/ZEN (n ¼ 3)
Wheat
GC/MS (mg/kg)
DON kita (mg/kg)
HPLC (mg/kg)
ZEN kita (mg/kg)
ICS test DON/ZEN (n ¼ 3)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
NDd 1.44 0.11 ND 0.13 2.87 0.35 0.18 ND 0.18 ND ND 0.17 0.10 0.34 0.27 ND ND 0.46 0.14 0.41 ND ND 0.56 1.16 1.09 2.57 2.90 2.35 2.09 2.50
ND 1.68
ND 8.6 12.6 23.1 15.8 ND ND ND 22.1 ND ND ND ND 14.7 21.5 23.3 ND ND ND ND ND ND ND 39.3 87.6 ND 141.7 323.0 415.1 402.3 469.3
ND 10.1 16.3 15.2 18.1 ND ND ND 24.2 ND ND ND ND 20.1 28.3 17.5 ND ND ND ND ND ND ND 32.8 100.2 ND 153.2 289.3 400.4 382.2 500.1
b/, /, / þc/, þ/, þ/ /, /, / /, /, / /, /, / þ/, þ/, þ/ /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / /, /, / þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ þ/þ, þ/þ, þ/þ
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.53 1.40 2.59 1.36 0.56 2.68 1.10 1.14 1.01 4.59 ND 1.15 2.90 1.80 2.45 2.12 2.10 1.27 1.58 1.01
1.66 1.57 2.63 1.22 0.47 2.89 1.03 1.05 1.11 4.46
38.2 ND 36.2 67.3 ND 31.2 18.3 36.2 19.4 ND ND 33.2 ND ND ND ND 27.2 48.3 ND ND
35.1 ND 45.1 73.1 ND 20.4 23.2 32.1 18.3 ND ND 21.3 ND ND ND ND 36.4 50.2 ND ND
þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/þ, þ/þ, þ/þ /, /, / þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ /, /, / þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ þ/, þ/, þ/ /, /, /
a b c d e
Screening by RidascreenÒ Fast DON or ZEN ELISA kits. Positive result, T1 or T2 line vanished. Negative result, both C line and T line ( T1 or T2) appeared clearly. Not detected (ND). Limit of determination of DON, 0.2 mg/kg.
DON and ZEN, respectively (Fig. 2(C)). So the detection limit of the ICS test for DON and ZEN was 100 and 6 ng/mL, respectively. 3.4. Stability of ICS After treatments of sample pad, conjugate pads and NC membrane as described above, they were laminated into a sheet of
plastic backing orderly and cut into test strip. The assembly completed ICS were kept at 4 C and room temperature for 1 year in plastic bags containing silica gel desiccant. During the storage, the ICS were tested with the DON/ZEN mixture solutions (0/0, 25/2, 100/6 ng/mL) at an interval of 7 days in the former 3 months, and then an interval of 30 days for the later 7 months. As shown in Fig. 3, the ICS stored at 4 C and room temperature for 1 year gave
Fig. 4. Simultaneous detection of DON and ZEN with ICS in 31 maize (A) and 20 wheat (B) samples.
12
Z.-B. Huang et al. / Food Control 28 (2012) 7e12
the same results at the test lines and C line, when the DON/ZEN mixture standards (0/0, 100/6, 100/0, and 0/6 ng/mL) were assayed. Therefore, the ICS developed in this study can be stored for 1 year at room temperature. The result indicated that the ICS may be suitable as a commercial kit. 3.5. Simultaneous detection of DON and ZEN in wheat and maize samples Thirty-one natural maize samples purchased in local markets and 20 wheat samples provided by Hebei Provincial State Grain and Oil Quality Inspection Station (China) were analyzed using the ICS test and then confirmed with ELISA and instrumental analysis (GC/ MS for DON and HPLC for ZEN determination). Results were shown in Table 1. DON positive results including sample 2, 6, 25, 27, 28, 29, 30 and 31 of maize, and sample 1, 2, 3, 4, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, and 19 of wheat containing range from 1.01 mg/kg to 4.59 mg/kg, in ELISA and GC/MS gave a positive result with one or two red line on the ICS test (Fig. 4). ZEN positive results including sample 25, 27, 28, 29, 30 and 31 of maize, and sample 4 of wheat containing rage from 67.3 mg/kg to 500.1 mg/kg. All the contained wheat and maize samples with DON levels lower than 1000 mg/kg, and ZEN levels lower than 60 mg/kg, indicating that they were DON and ZEN negative, respectively, in the ICS test. Results of samples analysis obtained from the ICS were in a good agreement with those obtained form ELISA and instrumental analysis. This results show that the three methods corresponded well, and the ICS test gave neither false-positive nor false-negative results. 4. Conclusions A rapid ICS test was developed to simultaneously detect DON and ZEN in wheat and maize samples. The ICS test can be used as qualitative tools for the rapid simultaneous screening of DON and ZEN contamination within 10 min (including rapid extraction and assay time) on-site. The ICS test had a visual detection limit of 100 ng/mL for DON and 6 ng/mL for ZEN. The sensitivities of the current assay methods were sufficient to detect DON and ZEN at the maximum residue limit of 1000 mg/kg and 60 mg/kg, respectively, proposed for legislation in China and are suitable for use as rapid simultaneous screening tests for DON and ZEN. Acknowledgments This work was financially supported by grants from the National High Technology Research and Development Program of China (863 Program, No. 2007AA10Z427), and the National Natural Science Foundation of China (31160308 and 31171696), and the Research Foundation for Young Scientists of State Key Laboratory of Food Science and Technology, Nanchang University, China (SKLFQN-201102). References Abbes, S., Quanes, Z., Salah-Abbes, J. B., Abdel-Wahhab, M. A., Oueslati, R., & Bacha, H. (2007). Preventive role of aluminosilicate clay against induction of micronuclei and chromosome aberrations in bonemarrow cells of Balb/C mice treated with zearalenone. Mutation Research, 21, 136e144. Abid-Essefi, S., Baudrimont, I., Hassen, W., Ouanes, Z., Mobio, T. A., Anane, R., et al. (2003). DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by vitamin E. Toxicology, 192, 237e248. Beyer, M., Verreet, J. A., & Ragab, S. M. (2005). Effect of relative humidity on germination of ascospores and macroconidia of Gibberella zeae and deoxynivalenol production. International Journal of Food Microbiology, 98, 233e240. Boussema-Ayed, I., Ouanes, Z., Bacha, H., & Abid, S. (2007). Toxicities induced in cultured cells exposed to zearalenone: apoptosis or mutagenesis? Journal of Biochemical and Molecular Toxicology, 21, 136e144.
Cetin, Y., & Bullerman, L. B. (2006). Confirmation of reduced toxicity of deoxynivalenol in extrusion-processed corn grits by the MTT bioassay. Journal of Agricultural Food Chemistry, 54, 1949e1955. Cho, Y. J., Lee, D. H., Kim, D. O., Min, W. K., Bong, K. T., Lee, G. G., et al. (2005). Production of a monoclonal antibody against ochratoxin A and its application to immunochromatographic assay. Journal of Agricultural and Food Chemistry, 53, 8447e8451. Delmulle, B. S., De Saeger, S. M. D. G., Sibanda, L., Barna-Vetro, I., & Van Peteghem, C. H. (2005). Development of an immunoassay based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. Journal of Agricultural and Food Chemistry, 53, 3364e3368. Desmarchelier, A., Oberson, J. M., Tella, P., Gremaud, E., Seefelder, W., & Mottier, P. (2010). Development and comparison of two multiresidue methods for the analysis of 17 mycotoxins in cereals by liquid chromatography electrospray ionization tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 58, 7510e7519. Ediage, E. N., Di Mavungu, J. D., Monbaliu, S., Van Peteghem, C., & De Saeger, S. (2011). A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples. Journal of Agricultural and Food Chemistry, 59, 5173e5180. Guo, Y. C., Ngom, B., Le, T., Jin, X., Wang, L. P., Shi, D. S., et al. (2010). Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle. Analytical Chemistry, 82, 7550e7555. Kolosova, A. Y., De Saeger, S., Sibanda, L., Verheijen, R., & Van Peteghem, C. (2007). Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of zearalenone and deoxynivalenol. Analytical and Bioanalytical Chemistry, 389, 2103e2107. Kolosova, A. Y., Sibanda, L., Dumoulin, F., Lewis, J., Duveiller, E., Van Peteghem, C., et al. (2008). Lateral-flow colloidal gold-based immunoassay for the rapid detection of deoxynivalenol with two indicator ranges. Anayltica Chimica Acta, 616, 235e244. Liu, B. H., Tsao, Z. J., Wang, J. J., & Yu, F. Y. (2008). Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip. Analytical Chemistry, 80, 7029e7035. Meneely, J., Fodey, T., Armstrong, L., Sulyok, M., Krska, R., & Elliott, C. (2010). Rapid surface plasmon resonance immunoassay for the determination of deoxynivalenol in wheat, wheat products, and maize-based baby food. Journal of Agricultural and Food Chemistry, 58, 8936e8941. Molinelli, A., Grossalber, K., Führer, M., Baumgartner, S., Sulyok, M., & Krska, R. (2008). Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat. Journal of Agricultural and Food Chemistry, 56, 2589e2594. Monbaliu, S., Van Poucke, C., Detavernier, C., Dumoulin, F., Van De Velde, M., Schoeters, E., et al. (2010). Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. Journal of Agricultural and Food Chemistry, 58, 66e71. Neuhof, T., Koch, M., Rasenko, T., & Nehls, I. (2008). Distribution of trichothecenes, zearalenone, and ergosterol in a fractionated wheat harvest lot. Journal of Agricultural and Food Chemistry, 56, 7566e7571. Nuryono, N., Noviandi, C. T., Böhm, J., & Razzazi-Fazeli, E. (2005). A limited survey of zearalenone in Indonesian maize-based food and feed by ELISA and high performance liquid chromatography. Food Control, 16, 65e71. Ouanes, Z., Abid, S., Ayed, I., Anane, R., Mobio, T., Creppy, E. E., et al. (2003). Induction of micronuclei by zearalenone in vero monkey kidney cells and in bone marrow cells of mice: protective effect of vitamin E. Mutation Research, 538, 63e70. Romero-Gonzalez, R., Martinez Vidal, J. L., Aguilera-Luiz, M. M., & Frenich, A. G. (2009). Application of conventional solid-phase extraction for multimycotoxin analysis in beers by ultrahigh-performance liquid chromatography-tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 57, 9385e9392. Rotter, B. A., Prelusky, D. B., & Pestka, J. J. (1996). Toxicology of deoxynivalenol (vomitoxin). Journal of Toxicology and Environmental Health, 48, 1e34. Saha, D., Acharya, D., Roy, D., Shrestha, D., & Dhar, T. K. (2007). Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples. Anayltica Chimica Acta, 584, 343e349. Schenzel, J., Schwarzenbach, R. P., & Bucheli, T. D. (2010). Multi-residue screening method to quantify mycotoxins in aqueous environmental samples. Journal of Agricultural and Food Chemistry, 58, 11207e11217. Shephard, G. S. (2008). Determination of mycotoxins in human food. Chemical Society Reviews, 37, 2468e2477. Shim, W. B., Dzantiev, B. B., Eremin, S. A., & Chung, D. H. (2009). One-step simultaneous immunochromatographic strip test for multianalysis of ochratoxin A and zearalenone. Journal of Microbiology and Biotechnology, 19, 83e92. Shim, W. B., Kim, K. Y., & Chung, D. H. (2009). Development and validation of a gold nanoparticle immunochromatographic assay (ICG) for the detection of zearalenone. Journal of Agricultural and Food Chemistry, 57, 4035e4041. Wang, S., Quan, Y., Lee, N., & Kennedy, I. R. (2006). Rapid determination of fumonisin B1 in food samples by enzyme-linked immunosorbent assay and colloidal gold immunoassay. Journal of Agricultural and Food Chemistry, 54, 2491e2495. Xu, Y., Huang, Z. B., He, Q. H., Deng, S. Z., Li, L. S., & Li, Y. P. (2010). Development of an immunochromatographic strip test for the rapid detection of deoxynivalenol in wheat and maize. Food Chemistry, 119, 834e839.