Article pubs.acs.org/JAFC

High Specific Monoclonal Antibody Production and Development of an ELISA Method for Monitoring T‑2 Toxin in Rice Yanshen Li,*,† Xiangshu Luo,‡ Shupeng Yang,‡ Xingyuan Cao,‡ Zhanhui Wang,‡ Weimin Shi,‡ and Suxia Zhang*,‡ †

College of Life Science, Yantai University, Yantai 264005, People’s Republic of China Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: This research produced a highly-specific and sensitive anti-T-2 toxin monoclonal antibody (mAb), and developed a rapid and sensitive competitive indirect enzyme-linked immunosorbent assay (ELISA) method for monitoring T-2 toxin in rice. The mAb showed a negligible cross-reactivity value (CR) to most of the mycotoxins, and it could specifically bind to T-2 toxin without other mycotoxins, including HT-2 toxin (CR value at 3.08%), which exhibited a similar structure to T-2 toxin. The limit of detection (LOD) value, measured by IC10, was 5.80 μg/kg. In spiked samples, mean recoveries ranged from 72.0% to 108.5% with intraday and interday variation less than 16.8 and 13.7%. This proposed protocol was significantly confirmed by a reliable ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method and significant correlation was obtained. KEYWORDS: T-2 toxin, high-specific monoclonal antibody, ELISA, rice

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animals.20 Some of its metabolites are equally or even more toxic than T-2 toxin.10 Once animals or human beings are exposed to T-2 toxin, there are no effective treatments for detoxication. In order to avoid the unnecessary exposure to T-2 toxin, it is necessary to develop rapid, sensitive, and accurate analytical methods for the detection of T-2 toxin. In the past, many investigations about T-2 toxin detection have been reported. From the structure, T-2 toxin showed weak UV absorption. And High-Performance Liquid Chromatography (HPLC) with Ultraviolet (UV) detector is not suitable for T-2 toxin detection while a fluorescence detector (FLD) provides high-sensitivity, selectivity, and repeatability after derivatization.21−23 Recently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is applied as a widespread technique for the simultaneous determination of T-2 toxin without derivatization.24−26 Also, the MS/MS spectrometry detector provides the most reliable results by satisfying the European Union’s technical criteria with four identification points.27 Gas chromatography applied MS/MS detector, flame ionization detector (FID), or FLD are also widely used as qualitative and quantitative technique for T-2 toxin determination.28−30 However, these instrumental analytical methods require precise and expensive equipment. And they cannot meet the rapid screening demands. For rapid detection, immunoassays with simplicity, low-cost, and high throughput advantages have been taken into consideration. Enzyme-linked immunosorbent

INTRODUCTION T-2 toxin, one of type A trichothecenes, is mainly produced by different Fusarium species, including F. acuinatum, F. poae, and F. soprotrichioides.1,2 These Fusariums often grow on cereal grains in inappropriate storage conditions, such as temperature, humidity, and different types of substrates.3,4 Surveys about the occurrences of T-2 toxin in grains, including barley, beans, maize, oats, soybean, wheat, and some other cereal-based products have been reported.5−7 The European Union (EU) reported that EU members were exposed to Fusarium toxins since 2003,8 and T-2 toxin was the highest toxic contaminant in cereals from EU member states. Due to its 12,13-epoxy ring, which is responsible for the toxicological activity, T-2 toxin is one of the most toxic mycotoxin of the trichothecene family.9 In addition, it was reported that the acetyl groups and the isovaleryl or 3′hydroxyisovaleryl groups were also the toxophores, which could induce cell apoptosis in the thymus.10 T-2 toxin could cause a series of toxic effects, including emesis, diarrhea, lethargy, weight loss, hemorrhage, inhibition of immunity, necrosis, damage of cartilaginous tissues, apoptosis, and even death.11,12 Recently, it was also reported that T-2 toxin could induce Alimentary Toxic Aleukia (ATA) and Kashin-Beck Disease (KBD).13 Moreover, T-2 toxin also reportedly could inhibit the synthesis of protein14,15 and nucleic acid.16,17 On the basis of the detection techniques of histology, in situ detection of fragmented DNA, DNA agarose gel electrophoresis, or by flow cytometry, reports about T-2-induced apoptosis of the immune system, gastrointestinal tissues, and fetal tissues have been reported in recent years.15,18,19 T-2 toxin is very stable in different environments, while it could be metabolized and eliminated after ingestion in © XXXX American Chemical Society

Received: September 17, 2013 Revised: January 20, 2014 Accepted: January 22, 2014

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dx.doi.org/10.1021/jf404818r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Figure 1. Synthesis of T-2 toxin immunogen and coating antigen. reagents were analytical grade obtained from Beijing Reagent Corp. (Beijing, China) Apparatus. 96-Well polystyrene microtiter wells were purchased from Costar (Costar Inc., Milpitas, CA, U.S.). Waters Acquity UPLC system, Acquity BEH C18 column (50 × 2.1 mm2 I.D., 1.7 μm particle size), and Micromass Quattro Premier XE triple quadrupole mass spectrometer were from Waters (MA, U.S.). Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, U.S.) was obtained for this study. Monoclonal Antibody (mAb) Production. The structure of T-2 toxin is shown in Figure 1. Hapten (3-HS-T-2) was prepared by esterification at the C3 position. Briefly, 10 mg of T-2 toxin was dissolved in 0.4 mL pyridine. Then 210 mg of succinic anhydride was added and stirred for 4 h in a boiling water bath. Dry pyridine and the residues were redissolved using 5 mL of chloroform. Five mL of deionized water was added for washing, and then the water was removed with centrifugation at 3000g for 5 min. The washing step was repeated 4 times. Then the chloroform was dried to obtain hapten 3HS-T-2 under a gentle stream of nitrogen at 60 °C. This hapten was used to prepare the immunogen (3-HS-T-2-BSA) and coating antigen (3-HS-T-2-OVA). Briefly, solution A was prepared by dissolving the hapten in 1 mL of DMF, while Solution B was prepared by adding 25 mg BSA (OVA) and 15 mg EDC in 5 mL PBS (0.1 M, pH 8.0). Then,solutions A and B were mixed and stirred for 24 h at room temperature. After that, the mixture was dialyzed in PBS (pH7.4, 0.01 M) for 3 d to obtain immunogen and coating antigen (the synthesis protocol shown in Figure 1). The preparation procedure for anti-T-2 toxin mAb and immunoglobulin were performed as previously described.34 Briefly, immunize ten female BALB/c mice subcutaneously with immunogen emulsified in Freund’s complete adjuvant (FCA). For booster immunization, each mouse was immunized subcutaneously with immunogen in Freund’s incomplete adjuvant (FICA) every four weeks for four times (immune procedure was shown in SI Table S1). In order to select the optimum mouse for the subsequent fusion, the serum of each mouse was collected and the titer was monitored by the ELISA method ten days after the last injection. The mouse was boost immunized with immnogen without adjuvants four days before fusion. The splenocytes were fused SP2/0 myelome cells with the help of PEG 1500, and the hybridomas were cultured in 96-well plates. Select and screen the culture supernatant to make sure the hybridomas which excreted the highest affinity anti-T-2 toxin mAb. The cultured

assay (ELISA) is a very mature and well-applied technology for T-2 toxin monitoring.29 For the screening of T-2 toxin, the ELISA method generally based on a competitive assay format by using a specific anti-T-2 toxin antibody. Moreover, compared with instrumental analytical methods, ELISA method does not need complicated sample preparation, and could be applied for T-2 toxin screening.31 Most antibodies used in the previously reported immunoassays cannot distinguish T-2 toxin and HT-2 toxin (a metabolite of T-2 toxin) in the ELISA method due to their similar structure (Supporting Information, SI, Figure S1).31−33 When an ELISA method gave a positive result, it was difficult to make sure whether the contaminants were T-2 toxin, HT-2 toxin, or both. For this reason, a specific anti-T-2 toxin monoclonal antibody (mAb) was produced and characterized, which could only bind to T-2 toxin with negligible CR value to most mycotoxins, including HT-2 toxin. Using this specific antibody, an ELISA method for the determination of T-2 toxin in rice was developed. And this developed protocol was confirmed by UPLC-MS/MS (ultrahigh performance liquid chromatography-tandem mass spectrometry). To the best of our knowledge, this is the first time that T-2 toxin specific antibody was produced, and this developed ELISA method could specially recognize T-2 toxin rather than the combination of T-2 toxin and HT-2 toxin.

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MATERIALS AND METHODS

Chemicals and Reagents. T-2 toxin, HT-2 toxin, 3-acetyldeoxynivalenol (3-AcDON), 15-acetyl-deoxynivalenol (15-AcDON), deoxynivalenol (DON), neosolaniol (NEO), and nivalenol (NIV) standard were obtained from Fermentek biotechnology, Israel. BSA (bovine serum albumin), DOM-1 (SI Figure S1), OVA (ovalbumin), T-2-triol, and T-2-tetraol were obtained from Sigma-Aldrich (St. Louis. MO, U.S.). Peroxidase-conjugated goat antimouse IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa, U.S.). Water was purified using a Milli-Q Synthesis system from Millipore (Bedford, MA, U.S.). HPLC grade acetonitrile was purchased from Dima Technology (Muskegon, MI, U.S.). Other B

dx.doi.org/10.1021/jf404818r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Figure 2. MS/MS spectrum of T-2 toxin [M + NH4]+. hybrioma cells (5−10 × 106 cell) were injected intraperitoneally into a mouse, and the anti-T-2 toxin mAb was prepared from the ascites fluid. Principle and the Procedure of ELISA. The ELISA procedure for T-2 toxin determination was performed previously described.31 3HS-T-2-OVA was performed as the coating antigen and the anti-T-2 toxin mAb working solution was prepared at a dilution of 1/6000 in PBS. Each well was individually analyzed, and the values were plotted to yield the calibration curve. Calibration curves were prepared at 11 concentration levels with the initial concentration at 10 000 ng/mL. The following concentrations were one-third of the previous in turn. Each concentration was run in triplicate, and standard curves were fitted with a four-parameter logistic equation by using Origin version 7.0 (OriginLab, Northampton, MA, U.S.). Sample Preparaton for ELISA Analysis. Two grams of rice samples were weighed into 50 mL polypropylene centrifuge tubes. After that, 20 mL of ethyl acetate was added for extraction. Then the mixture was vortexed for 3 min and centrifuged at 5000 rpm for 15 min at 4 °C. Ten milliliter aliquots of the supernatant of each sample were drawn to other centrifuge tubes and dried under a gentle stream of nitrogen at 60 °C. The residue was redissolved by 1 mL of 10% methanol and filtered through a 0.22 μm membrane. Fifty microliter aliquots of the supernatant of each sample were added to a 96-well plate for ELISA analysis. Accuracy and Precision. Negative rice samples, which were previously confirmed by UPLC-MS/MS analyses, were spiked with T2 toxin at concentrations 40, 200, and 1000 μg/kg. Analysis was performed according to the developed protocol with five replicates each (n = 5), and accuracy and precision were calculated on the basis of the standard curve. UPLC-MS/MS Method. Results obtained from the developed ELISA protocol were verified and confirmed by an UPLC-MS/MS method. An Acquity BEH C18 column was applied in a Waters Acquity UPLC system for T-2 toxin separation, with the oven temperature maintained at 30 °C. A Micromass Quattro Premier XE triple quadrupole mass spectrometer detector was coupled to the UPLC system via an ESI interface. The analysis was performed in positive ionization mode. The optimized chromatographic conditions were as follows: Mobile phase, 0.005 mM ammonia−water/ acetonitrile (40:60, v/v); isocratic elution with the flow rate, 0.3 mL/min; and injection volume, 10 μL. For monitoring T-2 toxin, Multiple Reaction Monitoring mode was adopted. Quadrupoles 1 and 2 were set to transmit the precursor

molecular ion ([M + NH4]+) of T-2 toxin (m/z 484) and the product ions, respectively. In this research, we choose m/z 484 as the precursor ion and m/z 214.9 and m/z 244.9 as the product ions (Figure 2).

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RESULTS AND DISCUSSION mAb Preparation and Characterization. In this research, the anti-T-2 toxin mAb produced in this research showed high titer with a low LOD value. In order to characterize the specificity of the mAb, another 9 mycotoxins (SI Figure S1) were determined by this developed ELISA protocol. The crossreactivity (CR) values were calculated using the following equation. CR(%) =

IC50 of T‐2 toxin × 100% IC50 of analogue

where IC50 is half maximal inhibitory concentration of a substance. The selectivity of T-2 toxin and its analogues is shown in Table 1. It can be concluded that mAb shows a negligible CR value to most of the mycotoxins investigated in this research, including HT-2 toxin. The CR value and IC50 value of HT-2 toxin was 3.08% and 717 ng/mL, respectively. Considering the negligible CR value for other mycotoxins, this developed mAb could specially recognize T-2 toxin. Table 1. IC50 and Cross-Reactivity (CR) Value of T-2 Toxin Analogues

C

no.

analogue

IC50 (ng/mL)

CR (%)

1 2 3 4 5 6 7 8 9 10

T-2 HT-2 T-2 triol T-2 tetraol NEO NIV DON DOM-1 3-AcDON 15-AcDON

22.09 717 14 000 >100 000 >100 000 >100 000 >100 000 >100 000 >100 000 >100 000

100 3.08