New Approach for Development of Sensitive and Environmentally

To evaluate cross-reactivity, other mycotoxin (OTA, ZEN, DON, and AFB1) and fumonisin analog (FB2) at concentrations from ..... Bennett , J. W.; Klich...
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New Approach for Development of Sensitive and Environmentally Friendly Immunoassay for Mycotoxin Fumonisin B1 Based on Using Peptide-MBP Fusion Protein as Substitute for Coating Antigen Yang Xu, Bo Chen, Qing-hua He,* Yu-Lou Qiu, Xing Liu, Zhen-yun He, and Zheng-ping Xiong State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China S Supporting Information *

ABSTRACT: Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP). The prepared peptide-MBP fusion protein are “clonable” homogeneous and FB1-free products and can be used as a coating antigen in the immunoassay. The half inhibition concentration of the quantitative immunoassay setup with fusion protein (F1-MBP and F15-MBP) was 2.15 ± 0.13 ng/mL and 1.26 ± 0.08 ng/ mL, respectively. The fusion protein (F1-MBP) was also used to develop a qualitative Elispot assay with a cutoff level of 2.5 ng/ mL, which was 10-fold more sensitive than that measured for chemically synthesized FB1−BSA conjugates based Elispot immunoassay. The peptide-MBP fusion protein not only can be prepared reproducibly as homogeneous and FB1-free products in a large-scale but also can contribute to the development of a highly sensitive immunoassay for analyzing FB1. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.

M

Immunoassay is based on specific interaction between antigen and antibody, for the small molecules, and the reliability and sensitivity of the assay are severely limited by the quality of antigen conjugates (coating antigen or competing antigen) and antibody.13 Aiming at improving the sensitivity of immunoassay for small molecules, many researchers have focused their efforts on developing natural (poly and monoclonal antibody) and artificial (aptamers, antibody fragment, and molecular imprinted polymers) antibodies.14−16 However, recent studies have shown that the quality of haptencarrier conjugates is essential to the development of sensitive and reliability immunoassays.17 Furthermore, in regard to the immunoassay for mycotoxins, the conventional mycotoxin conjugates were usually chemosynthesized by “trial and error”,

ycotoxins are secondary metabolites produced by fungi that are capable of causing acute or chronic disease in humans and other animals.1 The worldwide contamination of foods and feeds with mycotoxins is a significant problem.2 Currently, many countries have set legislative limits for mycotoxin, including aflatoxin B1(AFB1), ochratoxin A(OTA), zearalenone(ZEN), deoxynivalenol(DON), patulin(PAT), and fumonisin B1(FB1) in foodstuffs.3,4 A major way to eliminate mycotoxins from food and feedstuffs is to detect contaminated raw materials. To date, several methods are established for mycotoxin determination, including thin layer chromatography,5 high-performance liquid chromatography coupled to fluorescence, or tandem mass spectrometrics detection,6−9 and gas chromatography coupled with electron capture.10,11 On the other hand, immunochemical methods have also been developed and found widespread application as rapid screening method for mycotoxins because of their high specificity, sensitivity, facilitate sample preparation, and ease of use.12 © 2014 American Chemical Society

Received: June 3, 2014 Accepted: July 23, 2014 Published: July 23, 2014 8433

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Figure 1. Schematic presentation of the construction of expression plasmid for peptide-MBP fusion protein (FB1-conjugates mimetics).

used as a coating antigen in widely applied microplate-based immunoassay. For example, an indirect competitive Phage ELISA for AFB1 was established with mimotope of AFB1; the assay exhibited an IC50 value of 14 μg/kg in samples for AFB1, and the linear range is 4−24 μg/kg.31 He et al.32 used OTA mimotope which, selected from a second-generation phage, displayed a peptide library to develop a phage ELISA for OTA; the IC50 value of the assay was 0.04 ng/mL. The mimotope for OTA was also used to develop a qualitative dipstick immunoassay with a cutoff level of 1 ng/mL. As for the phage-based immunoassay, phages with filamentous nature are “unconventional” reagents which can infect Escherichia coli. In addition, the peptide linked to the phage particles leads to complex difficulties in measuring the phage displayed peptide and quality control.21 As another way, it may be necessary to chemically synthesize selected peptide sequences as free, soluble peptides. This allows precise control of peptide concentration, without the avidity artifacts associated with pentavalent display on phage. Liu el al.33 have developed a peptide ELISA using the mimotope peptide of FB1 conjugated with bovine serum albumin (BSA) as a coating antigen, and the IC50 value was 6.06 ng/mL with a linear range of detection of 1.77−20.73 ng/mL. In this study, based on mimotope of mycotoxin, using mycotoxin fumonisin B1 as a model hapten, selected peptides that bind to FB1-McAb were expressed as monovalent, soluable fusions to the maltose binding protein (MBP). The purified fusion protein can be prepared reproducibly as homogeneous and FB1-free products and be used as the coating antigen in the immunoassay for sensitive analyzing FB1. The approach to develop highly sensitive and environmentally friendly immunoassay for mycotoxins has not been reported to the best of our knowledge. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.

involving not only the handling of toxic mycotoxin which pose a threat to the environment and human health, but also the lotto-lot variation.18 The efficiency of chemical conjugation of mycotoxins to a carrier protein or an enzyme is low because such conjugation involves extensive modification and blocking stages and causes substantial bridge group interference and unwanted cross-reaction.19 The preparation of mycotoxin conjugates has become one of the main bottleneck problems of development of immunoassay for mycotoxin. Recently, research has been conducted to develop the substitutes for mycotoxin conjugates. One approach for doing this is via generation of anti-idiotype antibodies (AId or Ab2), which refer to an antibody raised against the variable region of an original antibody (Ab1). This technique has been used to prepare the AId against mycotoxin T-2,20 FB1,21 and AFB122 by monoclonal, polyclonal, or alpacas nanoantibody technology and have been applied to immunoassays for mycotoxin determination. However, the difficulties and time-consuming inherent in AId generation, especially in producing an array of different AId, from which the most suited ones could be chosen, largely restrict the application in mycotoxin detection.23 Another approach to replace the chemically synthesized mycotoxin conjugates is via the phage displayed peptide technology. Phage-displayed peptide (mimotope) has been shown to be an alternative to the molecular recognition element for various biological targets. Mimotope or epitope mimic is a peptide that will mimic the antibody binding site on the antigen and compete with the native antigen for binding.24 Phage-displayed peptides have been used in a number of applications, including epitope mapping,25 identifying peptide ligand,26 and defining the protein−protein interaction.27 Researchers have also used the technology to select mimotopes for mycotoxins, including ZEN,28 DON,29 OTA30 and were applied to the detection of mycotoxins in cereal samples. However, current research concerning the mimotope are usually based on phage forms that are used as competing antigens; there are few reports that a mimotope was directly 8434

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by isopropylthio-β-D-galactoside (IPTG).36 The periplasmic proteins were extracted by osmotic shock, as described previously. In the following, amylose resin affinity purification was carried out and analyzed by 12% SDS−PAGE.37 The purified fusion proteins were used as coating antigen in the subsequent immunoassay. Quantitative Immunoassay Established with PeptideMBP Fusion Protein. As for this assay, 100 μL of peptideMBP fusion protein (diluted in PBS, pH = 7.4) was added to microwells in a 96-well microplate (Costar, no. 42592) and incubated overnight at 4 °C. After blocking with 5% skim milk in PBS and washing with PBST, 50 μL of each serial concentration of FB1 (from 0.1 to 100 ng/mL diluted in PBS) or sample extract equally with FB1-McAb was added to the wells and mixed; the mixture was incubated at 37 °C for 40 min. Following washing three times with PBST, 100 μL of 1:2000 dilution of HRP-conjugated goat antimouse antibody was incubated in the wells at 37 °C for 30 min. Finally, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to the washed wells. The optical density at 450 nm was detected on a microplate reader (Thermo Scientific). To measure the optimized dilution of the immunoassay reagents, a check-board assay was conducted by using a different dilution of fusion proteins and FB1-McAb in advance. To compare the effect of peptide-MBP fusion protein with chemically synthesized coating antigen, FB1−BSA conjugates were also coated in microplate wells and tested as the abovedescribed immunoassay procedures. Qualitative Immunoassay Using the Peptide-MBP Fusion Protein. A qualitative Elispot immunoassay for FB1 was also developed with peptide-MBP fusion proteins. The procedure was performed as follows: a PVDF membrane (0.45 μm, 7 × 7 mm squares) was prepared and the fusion protein (diluted in PBS, pH = 7.4) was spotted onto the membrane as described previously12 and kept at 4 °C until use. After that, the qualitative assay was performed as follows: prepared membrane strips were soaked in various concentrations (0, 1, 2.5, 5, 10, and 25 ng/mL) of FB1 (dissolved in PBS) or sample extracts, and then containing the appropriate dilution of FB1-McAb, they were incubated for 15 min at room temperature. After washing three times with PBST, the membrane was incubated with HRP-conjugated goat antimouse antibody (1:2000) for 15 min and washed and incubated with TMB substrate for 2 min. The membrane was then rinsed with tap water, the color intensity of the FB1-positive control and sample test zones were visually compared to FB1-negative control (which has the most intense color). Validation of Fusion Protein-Based Immunoassay for FB1. Validation of the peptide-MBP fusion protein-based immunoassays for FB1 were carried out by evaluating crossreactivity with other mycotoxins and by the determination of the spiked samples and incurred samples. To evaluate cross-reactivity, other mycotoxin (OTA, ZEN, DON, and AFB1) and fumonisin analog (FB2) at concentrations from 0.1 to 1000 ng/mL were applied to fusion protein-based ELISA and Elispot immunoassay, respectively. To measure the recovery of FB1, maize samples were spiked with a known amount of FB1 at a final concentration of 0, 10, 50, 100, 500, and 1000 μg/kg to FB1-free samples. The sample extracts and dilution methods were performed as follows: 5 g of FB1-free maize samples confirmed by HPLC were mixed with 25 mL PBS and ultrasonically extracted for 15 min. After centrifugation, the supernatant was diluted 1:2 with PBS for

MATERIALS AND METHODS Chemicals and Reagents. Mycotoxin Fumonisin B1, Fumonisin B2, Deoxynivalenol, Ochratoxin A, Zearalenone, and Aflatoxin B1 were purchased from Sigma (St. Louis, MO). Ph.D.-12 Phage Display Peptide Library Kit, vector pMAL-pIII, restriction enzymes Acc65I, EagI, and amylose resin were purchased from New England Biolabs (Beverly, MA), and HRP-conjugated anti-M13 antibody was from GE Healthcare Inc. (Piscataway, NJ). Anti-FB1 antibodies (FB1-McAb), FB1− BSA conjugates were prepared in our laboratory.34 Fumonisin B1 ELISA kit was obtained from Shenzhen Lvshiyuan Biotechnology (Lvshiyuan, China). Polyvinylidene fluoride (PVDF) membranes (0.45 μm, 26.5 × 3.75 cm) were purchased from Millipore Co. (Bedford, MA). Taq polymerase, minibest DNA fragment purification kit and T4 DNA ligase were purchased from TaKaRa (Dalian, China). M13KE insert extension primer (5′-HOCAT GCC CGG GTA CCT TTC TAT TCT C-3′) and the −96g gIII sequencing primer (5′CCC TCA TAG TTA GCG TAA CG-3′) were synthesized by Invitrogen, China. The organic and inorganic chemicals used were of analytical reagent grade. Biopanning and Identification of FB1 Mimotopes. In our previous work, loop-constrained heptapeptides that mimic FB1 were obtained from a disulfide-constrained 7-mer peptide library.33 In this work, a linear 12-mer peptide library was biopanned against FB1-McAb in order to obtain various affinities of FB1 mimotope. Biopanning of FB1 mimotopes were carried out at competitive-binding conditions. Briefly, in the first round of panning, 2.0 × 1011 pfu phages (100 μL) from the 12-mer phage-displayed peptide library was added in a 5 μg/mL FB1-McAb-coated well and reacted at 37 °C for 60 min. After washing with PBST [0.01 M PBS (pH 7.4) + 0.1% (v/v %) Tween-20], 100 μL of elution buffer [0.2 M glycine-HCl (pH 2.2), 1 mg/mL BSA] was added and the elution mixture was rocked gently for 20 min at room temperature. The elutate was pipetted into a microcentrifuge tube and neutralized with 15 μL of 1 M Tris-HCl. In the second panning round, the well was coated with 100 μL of 1 μg/mL FB1-McAb. After 30 min of incubation at 37 °C, specific phages were eluted with 100 μL of 50 ng/mL FB1, which dissolved in PBS (0.01 M, pH 7.4). For the third round selection, 100 μL of 1 μg/mL FB1-McAb was coated and incubated with second panning round amplified eluted phages at 37 °C for 10 min; specific phages were eluted with 100 μL of 5 ng/mL FB1. Individual phage isolate from the elution was evaluated for FB1-McAb binding by phage ELISA and DNA sequencing, as described by He et al.32 Preparation and Characterization of Peptide-MBP Fusion Protein. Construction of Expression Plasmid for Peptide-MBP Fusion Protein. The schematic diagram of constructing expression plasmid is presented in Figure 1. Briefly, single-stranded phage DNA from the phage particle in which the FB1 mimotope was displayed was extracted and amplified using a M13KE insert extension primer and the −96g gIII sequencing primer by PCR as described previously.35 The amplified FB1 mimotope gene fragment was digested with Acc65 I and the Eag I restriction enzyme (37 °C for 5 h) and then ligated into pMAL-pIII, and next transformed to E. coli ER2738. Expression plasmid was extracted from the transformants and sequenced as described previously.36 Expression and Characterization of Peptide-MBP Fusion Proteins. The constructed expression plasmid prepared above was transformed to E. coli TB1 cells and fusion protein induced 8435

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Figure 2. Competitive inhibition curves of FB1 mimotope F1 and F15. Each value is the average of 3 experimental replicates ± standard deviation.

Figure 3. (A) Competitive inhibition curves of the ELISA for FB1 established with peptide-MBP fusion protein (F1-MBP and F15-MBP) and conventional FB1−BSA conjugates as coating antigens, respectively. Each value is the average of 3 experimental replicates ± standard deviation. (B) SDS−PAGE (coomassie brilliant blue staining); the Mr marker reside in lane M, and the affinity-purified peptide-MBP fusion protein (F1-MBP) is indicated with a black arrow in lane 1.

from the third round selection specific binding to FB1-McAb. The DNA sequencing results showed that the 19 indirect ELISA-positive phages were virtually 2 clones (F1 and F15), the inset peptide sequence was N-N-A-A-M-Y-S-E-M-A-T-D and T-T-L-Q-M-R-S-E-M-A-D-D, respectively. Analysis of the peptide sequences revealed a striking sequence convergence to a core motif X-X-X-X-M-X-S-E-M--A-X-D (X represents random amino acid residues). Two competitive inhibition curves were established with these two verified FB1 mimotopes (F1 and F15), which were used as competitors in the immunoassy. As Figure 2 shows, the IC50 for F1 and F15 exhibited in the phage indirect competitive ELISA were 0.422 ± 0.021 ng/mL and 0.875 ± 0.059 ng/mL, respectively, which is more sensitive than the previously reported FB1 mimotope (phage displayed disulfide-constrained 7-mer peptide)-based phage ELISA (its IC50 was 1.2 ng/mL).33

assay analysis and additional dilution steps were performed in the case of the content of FB1 in the sample was higher than the detection linearity range of the assay. As to the analysis of incurred samples, 60 random cereal and feedstuff samples collected from markets and feedstuff factories were prepared and analyzed as the above-described methods.

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RESULTS AND DISCUSSION FB1 Mimotope Biopanning and Characterization. Enrichment for phage-displayed peptides with affinity to FB1McAb was observed after competitive-binding elution and selected phages enriched from an initial 1.0 × 106 pfu to 3.4 × 108 pfu. Thirty individual phage clones (designated as from F1 to F30) from the third round selected phages were randomly isolated to infect E. coli ER2738 cells for amplification. Phage indirect competitive ELISA revealed 19 of 30 selected clones 8436

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Figure 4. Effects of (A) pH, (B) ionic strength, and (C) methanol on the performance of the peptide-MBP fusion protein (F1-MBP)-based ELISA assay. Each value is the average of 3 experiment replicates ± standard deviation.

Production and Identification of Peptide-MBP Fusion Protein. Phage single-stranded DNA fragment isolated from selected FB1 mimotopes (F1 and F15) were PCR amplified, respectively, and digested by EagI and Acc65I. As to the vector of pMAL-pIII, it was digested with the same enzymes and isolated by 8% agarose gel electrophoresis. Two expression plasmids of peptide-MBP fusion protein were constructed and confirmed by DNA sequencing, respectively (see Figure S1 of the Supporting Information).

Then, constructed expression plasmids were transformed into E. coli TB1 cells. Before peptide-MBP fusion proteins were expressed by IPTG inducing, an orthogonal experiment implemented to analyze the effect of IPTG concentration, induction temperature, and time were carried out and the results showed that the optimal fusion protein expression was induced with 0.4 mM IPTG at 25 °C for 16 h. During SDS− PAGE, peptide-MBP fusion protein (named as F1-MBP and F15-MBP) ran as a single band at the predicted molecular mass 8437

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optimum antibody time, when no FB1 are present was also analyzed; the antibody incubation time of 15 min was determined to be sufficient to achieve required color intensity for detection. Increasing the incubation time beyond 15 min did not change the spot color intensity. In order to visually evaluate the cutoff levels (the lowest concentration of FB1 able to achieve 100% inhibition and thus no spot was observed) of the F1-MBP-based Elispot assay, FB1 was analyzed at different concentrations (0, 1, 2.5, 5.0, 10, and 25 ng/mL). As present in Figure 5B, the intensity of the spots

(calculated as 44 KD for the 1:1 fusion of peptide and MBP protein) (Figure 3B). Quantitative Immunoassay for FB1 Using PeptideMBP Fusion Protein. The F1-MBP and F15-MBP prepared above were coated in microplate wells, and quantitative immunoassay for FB1 was performed. After the checkerboard assay, the reagents of FB1-McAb, F1-MBP, and F15-MBP with concentration of 1.25, 5.0, 5.0 μg/mL, respectively, were selected as the working conditions. The sensitivity of the method was determined, as shown in Figure 3A; the limit detection of immunoassay established with F1-MBP and F15MBP, estimated from the mean (plus 2 standard deviations) of the 10 blank samples, was 0.32 and 0.21 ng/mL, respectively, and IC50 of the assay was 2.15 ± 0.13 and 1.26 ± 0.08 ng/mL, respectively, which was 10-fold more sensitive than that measured for conventional FB1−BSA conjugate-based ELISA under the same reaction condition (its IC50 of assay was 21.39 ± 1.15 ng/mL, Figure 3A). Reproducibility and reliability of the F1-MBP- and F15-MBP-based assays were demonstrated by the coefficient variations (CVs) of 7.5% and 10.2% for intra-assay, respectively (n = 6). In accordance with the test of crossreactivity, no cross-reaction with mycotoxin AFB1, ZEN, DON, and OTA was found, and the cross-reaction with fumonisin analog (fumonisin B2) was 9.22%. It was reported that ionic strength and pH can substantially interfere with the immunoanalytical measurement of the analyte.38,39 In this assay, the influence of buffer ionic strength (from 5 to 50 mM) was investigated. The obtained results indicated that the IC50 values were almost equal in 5, 10, and 25 mM, but the value of maximum optical density was significantly decreased with ionic strength values beyond 25 mM (Figure 4A). At the optimal ionic strength, the influence of pH was evaluated between 5.0 and 9.0. The results showed that the IC50 and optical density values were almost equal at pH 5.0, 6.0, 7.4, and 8.0, while the inhibition curve of the assay turned straight, and optical density values markedly decreased at pH 9.0 (Figure 4B). Methanol is the most commonly used in the mycotoxin sample extraction. In order to evaluate the methanol effect, the concentrations of 0, 5%, 10%, 20%, 30%, and 40% methanolPBS were examined, respectively, in fusion protein-based ELISA. As shown in Figure 4C, there were slight differences among 0, and 5%; however, beyond 10% methanol interfered with the shape of inhibition curves strongly, which indicated that the methanol effect of the reaction between antigen and antibody coincides exactly with the results reported previously.31 In consideration of the fact that FB1 have good solubility in waters, and taking account of the IC50 and optical density values, the best perfomance was achieved at 10 mM PBS in pH 7.4 as the working solution to dilute the FB1 standard and the sample extract in the following peptide-MBP fusion protein-based immunoassay. Qualitative Immunoassay Based on Peptide-MBP Fusion Protein. For qualitative Elispot immunoassay, it was necessary to ensure that the spot intensities of the negative controls were high enough to be observable and low enough to allow differentiation between samples with low analyte concentration.12 For this purpose, a checkerboard-type assay was performed by using different dilutions of immunoreagents (F1-MBP and FB1-McAb) and different incubation times. The assay incubated with 100 μg/mL F1-MBP and 35 μg/mL of FB1-McAb was chosen to be the minium concentration of reagents for a clear and high enough spot intensity. The

Figure 5. Evaluation of the cutoff level (the smallest amount of fumonisin B1 able to achieve 100% inhibition and therefore no spot color development) of the Elispot assay assessed visually. PeptideMBP fusion protein (F1-MBP) and conventional FB1−BSA conjugates were applied to the assay as coating antigen and different FB1 levels (0, 1, and 2.5 ng/mL) were analyzed, respectively.

decreased with increasing FB1 concentration and no spot color development on the membrane were obtained at the FB1 standard concentration increase to 2.5 ng/mL, which was 10fold more sensitive than that measured for the chemosynthetic FB1−BSA conjugate-based Elispot immunoassay under the same reaction condition (its cutoff level is 25 ng/mL, Figure 5A). Validation Studies. The validations were performed by analyzing the spiked maize samples using peptide-MBP fusion protein (F1-MBP)-based ELISA and ELispot assays, respectively. As present in Table 1, maize samples spiked with 10− 1000 μg/kg FB1 exhibited recoveries from 83.10% to 115.14% Table 1. Recoveries of FB1 Added to Maize Samples in Determinations Performed by Peptide-MBP Fusion Protein (F1-MBP) Based ELISA and Elispot Immunoassaysa

FB1 added (μg/kg) 10 50 100 500 1000

F1-MBP-based ELISA (n = 3)

F1-MBP-based Elispot assay (n = 3c)

(μg/kg), mean ± SD

test zone

8.47 57.82 104.75 518.37 1113.81

± ± ± ± ±

0.14 2.31 3.22 5.45 10.42

−,a−,− +,b+,+ +,+,+ +,+,+ +,+,+

a

Negative, an obvious dot was observed, FB1 values < cutoff levels of Elispot assay. bPositive, no dot was observed, FB1 values > cutoff levels of Elispot assay. cThe n = 3 means the same experiment was performed 3 times, and each reading was performed by one person. 8438

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compared to the commercial ELISA kit. The results obtained from the three methods were in agreement with each other.

by ELISA. As for the Elispot assay, clear spots were observed for maize samples spiked with 10 μg/kg FB1, but no dots were obtained for maize spiked with 50, 100, and 1000 μg/kg FB1. These results demonstrated that adequate recovery and efficiency from cereals were achieved with peptide-MBP fusion protein-based immunoassay. A total of 60 samples of cereals and feedstuffs obtained from Chinese markets were analyzed using peptide-MBP fusion protein (F1-MBP)-based ELISA and ELispot assays, and commercial ELISA kit, respectively. Among the 60 samples, 15 of 35 maize samples, 7 of 20 feedstuffs were FB1 positive and 5 rice samples were all FB1 negative detected by F1-MBP-based ELISA (Table 2). The limits of detection (or cutoff level) of the

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CONCLUSIONS In this work, we described a new approach for development of sensitive and environmentally friendly immunoassay for mycotoxin. On the basis of the mimotope of mycotoxin FB1, selected peptides that bind to FB1-McAb were expressed as monovalent, soluable fusions to the maltose binding protein (MBP). The peptide-MBP fusion protein are “clonable” homogeneous products expressed from constructed express plasmid, which can prepare reproducibly in large scale and low cost. Peptide-MBP fusion protein provides practical specificity and reliability in the standard qualitative and quantitative immunoassay. Notably, the peptide-MBP fusion protein-based immunoassay is 10-fold more sensitive than that measured for the chemosynthetic FB1−BSA conjugate-based immunoassay. Furthermore, the novel concept might provide potential applications to a general method for the immunoassay of various toxic small molecules.

Table 2. Analysis of Incurred Samples by Peptide-MBP Fusion Protein (F1-MBP)-Based ELISA, Elispot Immunoassay and Commercial ELISA Kita sample maize

feedstuff

rice

number

F1-MBP-based ELISA (μg/kg, n = 3)

commercial ELISA kit (μg/kg, n = 3)

F1-MBP-based Elispot assay (μg/kg, n = 3f)

M1 M2 M4 M6 M7 M10 M12 M13 M15 M18 M19 M21 M22 M23 M26 F1 F3 F4 F7 F10 F12 F14 R1 R2 R3 R4 R5

420.5 ± 3.2 131.9 ± 8.3 46.8 ± 2.2 58.3 ± 4.3 189.7 ± 3.1 376.3 ± 9.5 130.9 ± 10.4 263.1 ± 9.6 1783.7 ± 34.5 650.9 ± 15.3 1289.3 ± 17.8 601.9 ± 18.4 711.9 ± 35.8 223.7 ± 16.5 96.3 ± 8.2 89.5 ± 3.0 15.1 ± 0.3 482.0 ± 5.2 35.1 ± 1.2 80.3 ± 4.6 43.2 ± 3.7 25.1 ± 2.4 ND ND ND ND ND

462.1 ± 4.2 211.9 ± 5.6 NDc ND 190.3 ± 3.9 354.2 ± 13.2 142.1 ± 11.3 239.8 ± 15.2 1617.8 ± 28.7 694.4 ± 17.1 1114.4 ± 54.8 805.3 ± 22.5 720.3 ± 25.6 244.7 ± 17.8 ND ND ND 451.2 ± 4.3 ND ND ND ND ND ND ND ND ND

+,b +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + +, +, + −d, −, − +, +, + +, +, + +, +, + +, +, + +, +, ±e −, −, − −, −, − −, −, − −, −, − −, −, −

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-791-8830 5177. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by grants from the National Natural Science Funds (Grants NSFC-31360386, NSFC-31201360, and NSFC-31171696), the Jiangxi Province Key Technology R & D Program (Grant 2014BBG70090), the Natural Science Foundation of Jiangxi, China (Grant 20132BAB214005), and by a grant of the Education Department of Jiangxi Province (Grant GJJ13095).

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REFERENCES

(1) Bennett, J. W.; Klich, M. Clin. Microbiol. Rev. 2003, 16, 497−516. (2) Zain, M. E. J. Saudi Chem. Soc. 2011, 15, 129−144. (3) Food and Agriculture Organization of the United Nations. Worldwide Regulations for Mycotoxins in Food and Feed in 2003. FAO Food and Nutrition Paper 81, Food Quality and Standards Service (ESNS): Rome, Italy, 2004. (4) European Commission Regulation.. Official Journal of the European Union 2006, L364, 5−24. (5) Larionova, D.; Goryacheva, I. Y.; Van Peteghem, C.; De Saeger, S. World Mycotoxin J. 2011, 4, 113−117. (6) Lerda, D.; Ambrosio, M.; Kunsagi, Z.; Stroka, J. J. AOAC Int. 2013, 96, 331−340. (7) Pascale, M.; Panzarini, G.; Powers, S.; Visconti, A. Food Analytical Methods 2014, 7, 555−562. (8) Zachariasova, M.; Lacina, O.; Malachova, A.; Kostelanska, M.; Poustka, J.; Godula, M.; Hajslova, J. Anal. Chim. Acta 2010, 662, 51− 61. (9) Soleimany, F.; Jinap, S.; Abas, F. Food Chem. 2012, 130, 1055− 1060. (10) Kong, W.; Zhang, X.; Shen, H.; Ou-Yang, Z.; Yang, M. Food Chem. 2012, 132, 574−581.

a

The limits of detection (or cutoff level) of the F1-MBP-based ELISA, commercial ELISA kit, and the F1-MBP-based Elispot assay was 10, 100, and 25 μg/kg, respectively. bPositive; no dot was observed. cNot detectable. dNegative; an obvious dot was observed. eNegative/ positive; an unclear dot was observed. fThe n = 3 means the same experiment was performed 3 times, and each reading was performed by one person.

three methods were 10, 100, 25 μg/kg, respectively. A total of 22 out of the 60 samples tested positive for FB1 by the F1MBP-based ELISA, as shown in Table 2. In addition, FB1 was detected in 21 samples by the F1-MBP-based ELispot assay and 13 samples by the commercial ELISA kit. Much more samples were determined by the peptide-MBP fusion protein-based immunoassay to be positive because of higher sensitivity 8439

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Analytical Chemistry

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dx.doi.org/10.1021/ac502037w | Anal. Chem. 2014, 86, 8433−8440