Journal of Food Safety ISSN 1745-4565

AN IMMUNOARRAY FOR THE SIMULTANEOUS DETECTION OF TWO MYCOTOXINS, OCHRATOXIN A AND FUMONISIN B1
jfs_314 408..416

XICHUN WANG1,2, HAIBIN ZHANG1,3, HAIMING LIU1, CHENGHUA HE1, AIHUA ZHANG1, JINRONG MA1, YANNA MA1, WENDA WU1 and HAO ZHENG1
1 2

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, China

Corresponding author. TEL: +86-25-84396478; FAX: +86-25-84396434; EMAIL: haibinzh@njau.edu.cn Accepted for Publication February 17, 2011 doi:10.1111/j.1745-4565.2011.00314.x

ABSTRACT
The two mycotoxins Ochratoxin A (OTA) and Fumonisin B1 (FB1) generate substantial global concern due to their toxicity in both humans and animals. Herein we describe the development of an immunoarray to detect these toxins. The immunoarray was prepared by immobilization of two antigen (Ag) conjugates on aldehydized slides. Standard curves were plotted, and the experimental conditions were optimized. The sensitivities were 5.43 ng/mL and 109.06 ng/mL, with detection ranges of 0.11 0 and 5200 ng/mL for OTA and FB1, respectively, with good logistic and linear correlation (R2 > 0.99). Next, unknown samples were detected by immunoarray and enzyme-linked immunosorbent assay (ELISA). The errors were relatively small for both methods, while the detection range of the immunoarrays was broader and more sensitive than traditional ELISA. The high-throughput immunoarray is a novel method for concurrent analysis of multiple mycotoxins that is technically simple, highly sensitive and relatively inexpensive.

PRACTICAL APPLICATIONS
The immunoarray technique used in this study provides an analytical tool with the specicity and sensitivity for rapid detection of two mycotoxins, Ochratoxin A and Fumonisin B1.

INTRODUCTION
Mycotoxins are secondary metabolites produced by various fungi which contaminate a large variety of foods, with toxic effects to animals and human. Ochratoxin A (OTA) is mainly produced by Aspergillus and Penicillium species (Simarro doorten et al. 2004; Ringot et al. 2006), and is a worldwide common contaminant of cereal grains (Duarte et al., 2010), coffee (Mantle 2000; Joosten et al. 2001), grapes (Sage et al. 2002), red wine (Visconti et al. 1999; Castellari et al. 2000) and beer (Medina et al. 2005). It is also found in human blood, urine and milk, as well as in animal blood and tissues (Creppy et al. 1995). OTA is nephrotoxic, hepatotoxic, teratogenic and immunotoxic to several species of animals, and cause kidney and liver tumors in mice and rats (Ringot et al. 2006). Moreover, OTA is suspected to be involved in the etiology of Balkan endemic nephropathy, a human chronic inter408

stitial nephropathy which is most often associated with urinary tract tumors (Pfohl-Leszkowicz et al. 2002). Fumonisin B1 (FB1) is primarily produced by Fusarium moniliforme. The natural occurrence of FB1 has been reported in many areas worldwide in maize and maize-based products (Paepens et al. 2004). FB1 induces equine leukoencephalomalacia (Wilson et al. 1992), pulmonary edema in pigs (Motelin et al. 1994), hepatotoxicity and hepatocarcinogenesis in rats (Gelderblom et al. 2001), hepatopathy and nephrosis in sheep (Colvin and Harrison 1992), and immunosuppression in poultry. Epidemiological evidence also indicates a possible correlation between human esophageal cancer and fumonisins (Yoshizawa et al. 1994; Pitt 2000; Marasas 2001). OTA and FB1 were classied by the International Agency for Research on Cancer as possible human carcinogens (group 2B) (International Agency for Research on Cancer [IARC] 1993, 2002).
Journal of Food Safety 31 (2011) 408416 2011 Wiley Periodicals, Inc.

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AN IMMUNOARRAY FOR DETECTING TWO MYCOTOXINS

Rapid and accurate detection of trace chemical targets and studies on interactions between bio-macromolecules and small molecules (Delaunay-Bertoncini et al. 2003; Liu et al. 2007) remain formidable challenges in analytical elds. Microarrays provide a highly sensitive and precise technique for obtaining information from biological samples (MacBeath and Schreiber 2000; Du et al. 2005). Unlike DNA arrays, which are based on nucleic acid hybridization on a surface-immobilized template, protein chips or arrays have different proteins imprinted on solid support (such as glass slides, silicon and gel, etc.) as probes to analyze interactions between different proteins (Chiem and Harrison 1998; De wildt et al. 2000). With high-throughput, miniaturization, minimal reagent, parallelism and automation, protein arrays are becoming increasingly important tools for protein interaction studies and diagnostics. Immunoarrays are based on the molecule-specic immunological recognition of antigens (Ags) or antibodies (Abs) by Abs or Ags immobilized on the surface of a solid slides. When complementary Ags or Abs reacts with the probes on the array, an array image of the uorescent intensity (FI) can be captured by a laser scanner and analyzed by computer software. Simultaneously, high-output, automatic, highsensitivity multi-analysis may be achieved. There is only one binding site for each small molecule, which means the competitive method should be used. However, detection of these materials in food and foodstuffs with protein arrays or immunoarrays is seldom reported. In recent years, the risks associated with mycotoxin contamination of foods and animal feed has promoted the development of analytical methods to detect these toxins reliably. Thin-layer chromatography (Rottinghaus et al. 1992; Santos and Vargas 2002), high-performance liquid chromatography (Paepens et al. 2005; Ghali et al. 2009), enzyme-linked immunosorbent assay (ELISA) (Wang et al. 2006; Erkekoglu et al. 2010) and electrochemical immunosensor (Abdul Kadir and Tothill 2010) have been used extensively for the detection of these materials. However, chromatography methods are expensive and time consuming, and require appropriate instrumentation and trained personnel. ELISA-based kits have become the most popular and useful screening tool, but are generally expensive and suffer from cross-reactivity, producing false positive results. Immunosensor techniques are a major development in screening methods for mycotoxin determination. The advantages of immunosensors over conventional ELISA methods are: increased sensitivity and decreased lower detection limit,, cost effectiveness and ease of use, decreased requirement for expensive reagents and the portability of the devices, which provide digital signal readout (Tothill 2001). In the present work, we explored the feasibility of detecting two mycotoxins with an immunoarray.
Journal of Food Safety 31 (2011) 408416 2011 Wiley Periodicals, Inc.

MATERIALS AND METHODS

Reagents and Instruments
OTA, FB1, bovine serum albumin (BSA), and ovalbumin (OVA) were purchased from Sigma (St. Louis, MO). Cy3conjugated goat anti-mouse IgG was acquired from Wuhan Boster Bio-Technology (Wuhan, China) and was diluted in 0.05% BSA to phosphate-buffered saline tween (0.01 M pH 7.4 PBS, 0.05% Tween-20). Polyethylene glycol 20,000 was purchased from Nanjing Zhuyan Biothechnology (Nanjing, China). The OTA and FB1 monoclonal antibodies (mAbs) were from mice, and were prepared by our laboratory; the mAb dilution solution was prepared by adding 0.1% BSA to phosphate buffered saline Tween (PBST). Phosphatebuffered saline solution (PBS; 0.01 M) was prepared in water using 8.0 g/L NaCl, 0.2 g/L KCl, 3.58 g/L Na2HPO412H2O and 0.27 g/L KH2PO4. The pH value was adjusted to 7.2 with concentrated NaOH. All other chemical reagents were of analytical grade. Puried water was obtained using a Milli-Q water system (Millipore, Bedford, MA). All experiments were performed at room temperature (25 2C). The molecular mass of OVA conjugates (OTA coupled OVA conjugate [OTA-OVA] and FB1 coupled OVA conjugate [FB1-OVA]) were obtained by Ultraex II MALDI-ToF/ ToF/MS (Bruker Daltonik GmbH, Bremen, Germany). Aldehydelized glass slides were purchased from Shanghai BaiO (Shanghai, China). A Pixsys 5500 SQ chip arrayer (Catesian Technologies, Irvine, CA) was employed for sample spotting. ScanArray Express and Quantaray 3.0 software (Packard Bioscience, Meriden, CT) were employed for image capture and data analysis.

Preparation and Identication of Ag Conjugates by Mass Spectroscopy

The OTA-OVA and FB1-OVA conjugates were synthesized by an active ester reaction (Liu et al. 2008) and glutaraldehyde reaction (Yu and Chu 1999). After synthesis and concentration, they were identied by mass spectroscopy. The accurate coupling ratios (the number of hapten molecules per molecules of protein) of the conjugates could be measured by mass spectroscopy and calculated as follows: Coupling ratio = (molecular massconjugate - molecular massOVA)/ molecular masshapten.

Validation of the Two Ag Conjugates on the Chip

The OTA-OVA and FB1-OVA Ag conjugates were diluted to 30 mg/mL and 40 mg/mL, respectively, in a solution of PBS and glycerol (8:2, v/v), and then spotted on the chip. Their complementary mAbs and Cy3-conjugated goat anti-mouse
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IgG secondary antibody were added, in turn, for the Ag-Ab competitive reactions. Meanwhile, in order to test the specicity of the two mAbs, blanks used as controls, i.e., OVA and deoxynivalenol (DON)-OVA, were spotted as well.

Comparison between Immunoarray and ELISA

Two different and unknown concentrations of real OTA and FB1 in wheat samples were detected by immunoarray and ELISA simultaneously. Each sample was tested three times. Based on the obtained FIs and the standard curves which were plotted as described above, the concentrations of the unknown samples were calculated. Values for known and unknown samples were compared.

Optimization of Experimental Conditions

The optimum experimental conditions were tested and selected. Optimization of the conditions for detection of OTA was interpreted in detail, and the conditions of FB1 was tested and selected with homogeneous procedures.The OTA Ag conjugate was diluted to different concentrations (20, 30, 40 and 60 mg/mL), and then spotted in a 4 4 array, 10 nL per dot. To expedite the analytical procedure,aldehydelized glass slide was divided into ve relatively small units. Each small unit was comprised of a 4 2 array. The conjugates were immobilized on the surface of the glass slide in an enclosed box at room temperature for 1 h, and then stored at 4C. In order to investigate the best xing conditions and blocking times on the slide,three xing conditions (37C for 1 h, 4C for 1 h or 4C overnight) and three blocking times (0.5, 1 or 2 h) were tested. After blocking with 0.1% BSA for 0.5 h at 37C, the chip was rinsed with puried water three times, and allowed to dry naturally. Next, 10 mL 30% methanol and 10 mL different OTA mAb concentrations were added to each unit, and the chip was incubated for 0.5, 1 or 2 h at 37C. Simultaneously, OTA mAb was diluted 400, 800, 1,600, 3,200 and added to each small unit, and put into the enclosed box for 0.5 h at 37C. After being removed, they were rinsed with PBST three times and puried water three times. Subsequently, Cy3conjugated goat anti-mouse IgG secondary antibody was diluted to 1, 2, 3 or 4 mg/mL, and then 10 mL was added to each small unit, and the chip placed in the enclosed box for 0.5 h at 37C. After being removed, the chips were rinsed and dried as before. Different concentrations of standard solutions were prepared in 30% methanol. The OTA was prepared at 0.1, 0.5, 1.25, 2.5, 5 or 10 ng/mL, and FB1 was prepared at 5, 10, 25, 50, 100 or 200 ng/mL.

RESULTS AND DISCUSSION

Conjugate Verication by Mass Spectroscopy
Verication of conjugate synthesis and estimation of the hapten/protein ratio are usually performed simultaneously (Zhang et al. 2007). Because of the very low molecular mass of OTA (403.8) and FB1 (721.8) and the single reactive site of these mycotoxins, it is very difcult to combine or immobilize them on the aldehydelized glass slides efciently and directly. Therefore, chemical modication of the two mycotoxins was necessary. OVA, the most common carrier protein, were selected for the preparation of the small Ag conjugates. By coupling the conjugates on the slides, the specic mAbs could be captured. The molecular mass of OTA-OVA (50,350.141) and FB1-OVA (51,928.612) were determined by mass spectroscopy (Fig. 1), and the coupling ratios were 13:1 and 9.6:1.

Validation of the Two Ag Conjugates by Chip

There are many active aldehyde radicals on the surface of commercial glass slides. A condensation reaction can be generated between aldehyde radicals on the slide and amino groups of proteins, which immobilizes the Ag conjugates on the slide. After mAb addition, specic Ag-Ab reactions were analyzed. Validation results (Fig. 2) showed that the two Ag conjugates specically combine with their corresponding Abs with higher binding efciency, resulting in strong FIs. OVA was used as the blank (control). DON-OVA was also spotted; the FIs for both these controls were relatively low, which demonstrates the high specicity of the protein microarray. Therefore, the antibody microarray can be used for a competitive reaction to detect small molecules.

Preparation of the Immunoarray

From top to bottom, Ag conjugates of OTA and FB1 were spotted in turn (Ag conjugate concentrations as described above). After spotting, the slide was incubated in an enclosed box overnight at 4C. Next, it was blocked in 0.1% BSA for 1 h and rinsed with ultrapure water. The two mAbs were added as follows: Fig. 6a, OTA mAb; Fig. 6b, FB1 mAb; Fig. 6c, OTA mAb and FB1 mAb; Fig. 6d, OTA (5 ng/mL) and FB1 (100 ng/ mL) with the OTA and FB1 mAbs were added as well (mAb concentrations as described above). The slide was incubated in an enclosed box for 1 h at 37C again for the specic Ag-Ab competitive reactions. Then, 2 mg/mL Cy3-conjugated goat anti-mouse IgG secondary antibody was added, and the slide put in the enclosed box for 0.5 h at 37C.
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Optimization of Reaction Conditions

Proper reaction conditions are crucial for strong FIs, with sensitive and specic results. The acquired FIs reached saturation when OTA-OVA was diluted to 30 mg/mL, and OTA mAb was diluted 1,600 (FI 30,00035,000: Table 1). Meanwhile, 40 mg/mL Ag conjugate and 1,200 FB1 mAb were selected as the best conditions (Table 2).
Journal of Food Safety 31 (2011) 408416 2011 Wiley Periodicals, Inc.

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AN IMMUNOARRAY FOR DETECTING TWO MYCOTOXINS

FIG. 2. SCANS OF CONTROL (OVA), AND DON-OVA TO VALIDATE THE TWO ANTIGEN CONJUGATES (OTA-OVA AND FB1-OVA) The concentrations of OTA-OVA and FB1-OVA were 30 mg/mL and 40 mg/mL, respectively. The added OTA and FB1 mAbs were diluted 1,600 and 1,200, and the secondary antibody was added at a concentration of 2 mg/mL. FB1, Fumonisin B1; OTA, Ochratoxin A; OVA, ovalbumin.

TABLE 1. THE OTA-OVA FLUORESCENCE INTENSITY OTA mAb OTA-OVA (mg/mL) FIG. 1. MASS SPECTROGRAMS OF THE TWO MYCOTOXIN CONJUGATES (a) For OTA-OVA, the MW was 50,350.141, as determined by mass spectroscopy, and the coupling ratio was 13:1; (b) For FB1-OVA, the MW was 51,928.612, determined by mass spectroscopy, and the coupling ratio was 9.6:1. FB1, Fumonisin B1; OTA, Ochratoxin A; OVA, ovalbumin. 20 30 40 60 1/400 32,222 39,300 40,923 39,888 1/800 28,107 36,447 37,948 36,953 1/1600 25,573 33,918 35,764 32,125 1/3200 20,749 29,706 32,390 29,339

The FIs for 30 mg/mL and 60 mg/mL OTA-OVA were very similar; 30 mg/mL was selected as the optimum OTA-OVA concentration. OTA mAb diluted 1,600 was selected (uorescence intensity 30,00035,000), and the secondary antibody was added at 2 mg/mL concentration. FI, uorescence intensities; OTA, Ochratoxin A; OVA, ovalbumin.

These data show that the FIs increase as the Cy3conjugated goat anti-mouse IgG secondary antibody concentration increases. However, lower secondary antibody concentration may narrow the detection range; higher secondary antibody concentration would increase the expense. Therefore, 2 mg/mL Cy3-conjugated goat anti-mouse IgG secondary antibody was selected (FI 30,00035,000; Fig. 3). The highest signal to noise (S/N) ratio was obtained when the xed condition of Ag conjugates were xed at 4C overnight and blocked in 1% BSA blocking for 1h at 37C (Fig. 4).
Journal of Food Safety 31 (2011) 408416 2011 Wiley Periodicals, Inc.

Lower S/N ratios or high background was related to inappropriate xing conditions and blocking time. Overnight incubation with 4C may allow sufcient condensation reaction, and blocking for 1 h can block nonreactive sites completely. Furthermore, when OTA-OVA reacted with OTA-mAb for 1 h, we obtained the highest FI (34,219), but there was no statistically signicant increase at 2 h (Fig. 4). Subsequent Ag-Ab reactions were incubated for 1 h. Prolonging the Ag-Ab reaction beyond 2 h might decrease Ab activity, but it
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TABLE 2. THE FB1-OVA FLUORESCENCE INTENSITY FB1 mAb FB1-OVA (mg/mL) 20 30 40 60 1/400 25,176 32,708 47,659 46,551 1/800 21,102 30,557 41,050 38,784 1/1200 15,945 26,232 34,730 36,338 1/1600 11,660 13,545 19,678 23,033

The FIs for 40 mg/mL and 60 mg/mL FB1-OVA were very similar; 40 mg/mL was selected as the optimum concentration. FB1 mAb diluted 1,200 was selected (uorescence intensity 30,00035,000), and the secondary antibody was added at 2 mg/mL concentration. FB1, Fumonisin B1; FI, uorescence intensities; OTA, Ochratoxin A; OVA, ovalbumin.

FIG. 3. RELATIONSHIP BETWEEN CY3-CONJUGATED GOAT ANTI-MOUSE IgG SECONDARY ANTIBODY CONCENTRATION AND FLUORESCENCE INTENSITY Bars 14 represent different concentration of goat anti-mouse IgG-CY3 secondary antibody relative to the uorescence intensity.

did not decrease within 2 h. The xing conditions and blocking time of FB1-OVA, and the Ag-Ab incubation time, were similar to OTA.

Immunoarray for Mycotoxins

Simultaneous detection of both mycotoxins could be performed with an immunoarray. Different concentrations of OTA and FB1 were added for the indirect competitive reaction, and the standard curves for the two mycotoxins were plotted (Fig. 5). The results show that simultaneous and quantitative determination of both mycotoxins was possible with the array. FIs decreased by increasing the concentration of the mycotoxins standard. The detection ranges were 0.1 10 ng/mL (OTA) and 5200 ng/mL (FB1) with both logistic and linear correlation. All the determination coefcients (R2) were greater than 0.99, demonstrating good correlation between the target mycotoxins and the FIs. Concentrations lower than 0.1 ng/mL (OTA) and 5 ng/mL (FB1), and higher than 10 ng/mL (OTA) and 200 ng/mL (FB1) were analyzed,
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FIG. 4. OPTIMIZATION OF DIFFERENT REACTION CONDITIONS Relationship between the incubation conditions of the OTA-OVA conjugate to the S/N ratio to optimize the protocol. (A) xed at 37C for 1 h (a), at 4C for 1 h (b), or at 4C overnight (c); (B) blocked for 0.5 h (a), 1 h (b), or 2 h (c); and (C) mAb immobilization for 0.5 h (a), 1 h (b) or 2 h (c). Ag, antigen; Ab, antibodies; OTA, Ochratoxin A; OVA, ovalbumin.

and they deviated from the standard curve. So the limit of quantitation (LOQ) of OTA and FB1 in the method was in accord with their detection ranges. Because of the variance of inter-arrays and inevitable errors, repetition and stability are still major problems for immunoarrays, requiring further optimization. For this reason, standard curves must be plotted each time when the immunoarray is used. In order to test the specicity of the immunoarray, it was exposed to a mixture of Abs. Figure 6a,b show that there was no cross-reaction between OTA and FB1. In Fig. 6c, after the OTA and FB1 mAbs reacted with their complementary Ag conjugates spotted on the slide, the addition of Cy3-conjugated goat anti-mouse IgG secondary antibody
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AN IMMUNOARRAY FOR DETECTING TWO MYCOTOXINS

Comparison between Immunoarrays and ELISA for Detecting Mycotoxins

As competitive ELISA is a well-established and widely used method for quantitating small molecules, it was used to detect real samples to conrm the results of the immunoarray (Table 3). The relative standard deviations were between 5.629.56% (for immunoarray) and 8.299.84% (for ELISA) compared with the real values, which are relatively small and within both detection ranges. However, the detection ranges of immunoarrays are much broader and more sensitive than the traditional ELISA, especially for the detection of the two mycotoxins. Moreover, with the multiplex capacity of immunoarrays, they will be more useful in analysis conducted in the eld.

CONCLUSIONS
Our present work demonstrated that immunoarrays can simultaneously detect two mycotoxins, OTA and FB1, with a sensitivity (IC50) of 5.43 ng/mL (OTA) and 109.06 ng/mL (FB1), and detection ranges between 0.110 ng/mL (OTA) and 5200 ng/mL (FB1) with good logistic and linear correlation (R2 > 0.99). However, the repeatability and stability of immunoarrays must be optimized, and more mycotoxins should be integrated in the arrays in further studies. With the development of processing and preparation technologies and access to high-quality Abs, and as a sensitive, rapid and highthroughput method, immunoarray will have an important role in the detection of various mycotoxins in agricultural products and foodstuffs.

FIG. 5. IMMUNOARRAY STANDARD CURVES FOR THE TWO MYCOTOXINS (a) Standard curve for OTA, at nal concentrations of 0.1, 0.5, 1.25, 2.5, 5 and 10 ng/mL; (b) Standard curve for FB1, at nal concentrations of 5, 10, 25, 50, 100 and 200 ng/mL. FB1, Fumonisin B1; OTA, Ochratoxin A.

produced strong FIs. In Fig. 6d, after the addition of the mixture of OTA (5 ng/mL) and FB1 (100 ng/mL) mAbs, the FIs were sharply decreased. The most crucial factors for the OTA and FB1 mAbs used in immunoassays are good bioactivity and specicity. Phage display technology (Hust and Dubel 2004; Lensen et al. 2006) and ribosome display technology (Dufner et al. 2006) are alternative tools for generating high-quality Abs. Abs with higher titer and specicity, especially mAbs, can signicantly increase the accuracy and sensitivity of the immunoarray.

ACKNOWLEDGMENTS
This study was nancially supported by the National High Technology Research and Development Program of China (2007AA10Z429).

TABLE 3. SAMPLE DETECTION FOR TWO MYCOTOXINS BY IMMUNOARRAY AND ELISA Available MFI ( X s, n = 3) 32,278.16 216.59 27,210.35 168.46 18,733.28 145.34 30,828.00 229.75 15,507.19 164.83 10,660.01 117.13 Found by immunoarray (ng/mL) 0.46 2.11 4.87 9.86 113.47 139.25 Found by ELISA ( X s, n = 3, ng/mL) 120.36 8.28 158.12 9.19 Real (ng/mL) 0.5 2 5 10 100 150 RSD by immunoarray (%) 5.62 7.86 8.09 7.53 8.47 9.56 RSD by ELISA (%) 9.84 8.29

Samples 1OTA 2OTA 3OTA 4FB1 5FB1 6FB1

Refers to undetectable concentrations or no results. FB1, Fumonisin B1; OTA, Ochratoxin A.

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FIG. 6. SIMULTANEOUS DETECTION OF BOTH MYCOTOXINS Images of the chips after addition of Abs as follows: (a) OTA mAb; (b) FB1 mAb; (c) OTA and FB1 mAb; (d) mixture of OTA (5 ng/mL) and FB1 (100 ng/mL), and then the of OTA and FB1 mAbs were added. FB1, Fumonisin B1; OTA, Ochratoxin A; OVA, ovalbumin.

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