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Ochratoxin A Mimotope from Second-Generation Peptide Library and Its Application in Immunoassay Zhen-yun He, Qing-hua He,* Yang Xu,* Yan-ping Li, Xing Liu, Bo Chen, Da Lei, and Cheng-hao Sun State Key Laboratory of Food Science and Technology, Sino-Germany Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China ABSTRACT: With the advantage of replacing mycotoxins and their conjugates, mimotopes have been applied to immunoassays, the most common of which were seleted from random phage displayed peptide libraries. However, these mimotopes were limited by the diversities of the peptide libraries. The aim of this study was to demonstrate that a variety of mimotopes can be obtained by constructing a second-generation peptide library. Using mycotoxin ochratoxin A as a model system, a dodecapeptide mimotope was isolated after panning the second-generation peptide library. The half inhibition concentration of the chemiluminescent enzymelinked immunosorbent assay setup with this mimotope was 0.04 ng/mL, and the linear range was 0.006−0.245 ng/mL. The mimotope was also used to develop a qualitative dipstick assay with a cutoff level of 1 ng/mL. The method not only presents a high sensitivity but also contributes to the development of mimotope-based assays for mycotoxins avoiding the need of synthesizing toxic mycotoxin conjugates.

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0.08 ng/mL and a linear range of 0.02−0.3 ng/mL.10 However, they are limited to special instruments and skilled personnel and unsuitable for on-site screening. As a consequence, various kinds of immunochromatographic assays (ICG) have been developed for the on-site detection of OTA with cutoff levels range from 5 to 500 ng/mL.12−14 Mycotoxin used in both immunoassay and immunochromatographic assay might be toxic to human beings.19 To overcome this problem, several researchs have been conducted to develop efficient materials to replace mycotoxin. As early as in 1990s, anti-idiotype antibodies have been developed as toxin mimics that mimic the binding of fumonisin B1, deoxynivalenol, and trichothecene mycotoxin T-2.15−17 It was reported that an antiidiotype antibody had a wide cross-reactivity toward fumonisin B1 and B2, which would achieve simultaneous multiresidue determination of analogues.15 Nevertheless, anti-idiotype antibodies have not had real applications in mycotoxin immunoassays because they are difficult and expensive to produce. Another alternative develops rapidly in mycotoxin immunoassays is mimotope. By biopanning against phage display random peptide libraries, mimotopes were obtained and used as candidates for mycotoxins.18,19 An immunoassay for OTA avoiding the use of mycotoxin was established with mimotope, and the limit of detection was 150 pg/mL.20 Lai et al. used mimotope peptide to develop a colloidal gold strip to detect OTA from cereals, of which the detection limit was 10 ng/

chratoxin A (OTA) mainly produced by Penicillium and Aspergillus is a fungal toxin which is often found as a contaminant in a variety of foods and feedstuffs.1 A number of researches have revealed that OTA was the pathogen in nephropathies and urinary tract tumors.2 International Agency for Research on Cancer has classified OTA within Group 2B as a possible human carcinogen.3 Legal limits of OTA have been set in cereals at 5 μg/kg in the European Union (EU), Russia, China and as the same in the Codex Alimentarius Standard; the EU has also set a limit for OTA as 10 μg/kg in instant coffee and 2 μg/kg in grape juice.4−6 In order to minimize the risk of OTA to animals and humans, many studies have been performed to develop methods for OTA determination including instrumental analysis, immunoassay, and immunochromatographic assay (ICG). Instrumental techniques have been widely used for good accuracy and reproducibility, such as high-performance liquid chromatography (HPLC) and liquid chromatography− tandem mass spectrometry (LC−MS/MS). The limit of quantification for OTA was as low as 0.05 μg/kg in Portuguese rice and 0.01 ng/mL in urine.7,8 Nevertheless, instrumental method is time-consuming and expensive for sample preparation and analysis. Alternatively, immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization immunoassay (FPIA), are generally used to screen a mass of samples within a relatively short time.9−11 A FPIA with the detection limit of 0.7 ng/mL was established for rapid screening of OTA in red wine.11 Previously, an ultrasensitive chemiluminescent ELISA using anti-OTA monoclonal antibody was developed with a half inhibition concentration (IC50) of © 2013 American Chemical Society

Received: July 12, 2013 Accepted: September 16, 2013 Published: September 18, 2013 10304

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mL.21 As a living organism, phage-displayed mimotope appears easy to produce and has a relatively low cost. However these mimotopes were only selected from random peptide libraries, and the binding affinities to their corresponding antibodies were limited by the diversity of phage displayed peptide libraries and thus limited their application in immunological analysis. To generate phage displayed peptides with higher binding affinities to given targets, designing and panning a secondgeneration library is proven feasible.22,23 Some protocols such as oligonucleotide-directed random mutation, DNA shuffling, and error-prone PCR were proposed to construct a secondgeneration library.24−26 Affinity maturation is commonly used to improve the properties of antibodies selected from antibody libraries in vitro.26 Furthermore, this method was also applied to mimotope,27 antagonist,28 and agonist.23 It has been reported in the literature that polysaccharide capsule mimotope could be selected from the evolutionary phage peptide library for high affinity.27 By constructing and screening a B lymphocyte stimulator (BLyS) affinity maturation library, in which consensus residues flanking the motif sequence were seeded, the affinity of peptides that bind BLyS was raised approximately 100-fold.28 It has not been reported that the approach was used to develop the affinity and specificity of small molecules such as a mycotoxin involved mimotope to the best of our knowledge. In this study, a second-generation peptide library was constructed to obtain various affinities of OTA mimotopes. Mimotopes that were more suitable for immunoassay were selected to develop sensitive phage chemiluminescent ELISA and a polyvinylidene fluoride (PVDF) membrane based noninstrumental dipstick assay. This indicates the utility of the mimotope as a competitive antigen in OTA immunoassay.

pfu phages from the random peptide library were incubated in the OTA-McAb coated well for 10−60 min. Then unbound phages were washed with PBST (PBS containing 0.05% tween). Bound phages were eluted with elution buffer [0.2 M glycineHCl (pH 2.2), 1 mg/mL BSA] and neutralized with 1 M TrisHCl (pH 9.1). Finally, eluted phages were titered and amplified for the next round.29 After three rounds of screening, individual phage plaques from the third round eluted pool were amplified and purified for identification by phage ELISA.29 After being verified to be able to specifically bind to OTA-McAb, ELISApositive phage were used for single-stranded DNA islolation.30 Subsequently, the displayed peptides of corresponding phages were sequenced according to the manual. Second-Generation Peptide Library Construction. After OTA mimotopes selection in random peptides library, a second-generation peptide library was constructed aiming to develop a more sensitive mimotope-based immunoassay for OTA. The schematic diagram of construction of the secondgeneration peptide library was presented in Figure 1. The motif

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MATERIALS AND METHODS Chemicals and Reagents. The Ph.D. peptide display cloning system and Ph.D. phage display peptide library were purchased from New England Biolabs, Inc. (Beverly, MA). Anti-OTA monoclonal antibodies (OTA-McAb) were prepared in our laboratory. Mycotoxin ochratoxin A, aflatoxin B1, aflatoxin M1, citrinin, bovine serum albumin (BSA), ovalbumin (OVA), and horse radish peroxidase (HRP)-conjugated goat antimouse antibody were obtained from Sigma (St. Louis, MO). HRP-conjugated anti-M13 antibodies were purchased from GE Healthcare (Piscataway, NJ). 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate were purchased from Bio Basic Inc. (Toronto, Ontario). SuperSignal ELISA Pico chemiluminescent substrate was obtained from Thermo Scientific (Waltham, MA). PVDF membranes were purchased from Millipore Co. (Bedford, MA). The commercial ELISA kit (RIDASCREEN FAST Ochratoxin A) and ochratoxin cleanup columns were purchased from R-Biopharm AG (Darmstadt, Germany). The organic and inorganic chemicals used were of reagent grade. OTA Mimotopes Selection in Random Peptide Libraries. In our previous work, heptapeptides that mimic OTA were obtained from a linear 7-mer peptide library.20 In this work, a linear 12-mer peptide library (Ph.D.-12) and a disulfide-constrained 7-mer library (Ph.D.-C7C) were used to select dodecapeptides and cyclic heptapeptides that mimic the binding of OTA, respectively. A biopanning experiment was conducted as described by Liu et al.20 Briefly, a well of ELISA plate was coated with OTA-McAb in 0.01 M PBS (pH 7.4) overnight at 4 °C. After incubation at 4 °C for 2 h with blocking buffer 3% (w/v) BSA in PBS, approximately 1 × 1011

Figure 1. Schematic presentation of the cloning of randomized inserts into the phage vector; (a) annealing and extension; (b) double digestion, followed by DNA-ligation with M13KE in digested form. Nucleotide and the primer sequence used for synthesizing randomized inserts is shown: Cut site of restriction endonuclease EagI is lowercase and underlined. Cut sites of KpnI are lowercase and in bold. The FQLH motif is indicated in bold and uppercase, and the three random amino acids on either side of this motif. M = A + C, N = A + G + C + T.

sequence based on the selected dodecapeptides and cyclic heptapeptides above was incorporated into the design of the second-generation peptide library. The procedures were described as follows. First, a chemosynthetic 89-bp oligonucleotide (L12) randomized at both sides of the motif was annealed (heat to 95 °C and cool slowly to less than 37 °C) with a extension primer. After that, it was converted to double strand by extension (10 min, 37 °C; 15 min, 65 °C) with Klenow fragment (NEB). Next, the extension product was digested (3 h, 37 °C) with KpnI and EagI. Afer the digested duplex was purified by polyacrylamide gel electrophoresis (PAGE), it was ligated into a digested M13KE overnight at 16 °C. Afterward, the ligation product purified by ethanol precipitation was transfected into electrocompetent ER2738 cells by electroporation (25 μF, 200 Ω, 2.5 kV). At last, the transfected cells 10305

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were resuscitated, titered and amplified.29 To characterize the diversity of the final phage library, 50 phage clones were randomly picked up for DNA sequencing. Repanning of OTA Mimotopes in Second-Generation Peptide Library. Repanning of OTA mimotopes were perfomed through the second-generation library at competitive-binding condition. In the first round of panning, phages (1 × 1011 pfu diluted in 100 μL of PBS) from the evolved library was added in a 10 μg/mL OTA-McAb coated well and reacted at room temperature for 30 min. After washing with PBST and competition with 100 μL of 20 ng/mL OTA standard, specific binding phages were eluted and amplified. In the second panning round, the well was coated with 100 μL of 1 μg/mL OTA-McAb. After 10 min of incubation at room temperature, specific phages were eluted with 100 μL of 2 ng/mL OTA. For the third round selection, 1 μg/mL of 100 μL OTA-McAb was coated in a well; phages were eluted with 0.2 ng/mL OTA prior to a 5-min incubation. Individual phage isolates from the elutions were evaluated for OTA-McAb binding by phage ELISA and DNA sequencing as essentially the same as described above. Phage Chemiluminescent ELISA. A chemiluminescent ELISA for OTA was developed with an OTA mimotope isloated from the second-generation peptide library. The procedure was performed as follows: OTA-McAb was coated in microplate wells overnight at 4 °C and blocked for 1 h at 37 °C. The microplate was washed 3 times with PBST, then 50 μL of selected phages (OTA mimotopes obtained from secondgeneratin library) and 50 μL OTA at various concentrations (0, 0.001, 0.005, 0.025, 0.100, 0.500, 2.500, 10.000 ng/mL) were added to the respective wells for 10 min at 37 °C. After washing, 100 μL of 1:5000 dilution of HRP-conjugated antiM13 antibody was incubated in the wells for 30 min at 37 °C. Finally, 100 μL of chemiluminescent substrate was added to the washed wells. Chemiluminescence intensity was detected on a luminescence reader (Thermo Scientific). To measure the optimized dilution of the immunoassay reagents, a checkerboard assay was conducted by using different dilutions of phages and OTA-McAb in advance. Binding percentages were calculated as follows: binding (%) = A/A0 × 100%, herein A was the intensity in the presence of OTA and A0 was the intensity in their absence. Phage Dipstick Assay. The procedures were carried out as described previously.31 Briefly, a 0.45 μm PVDF membrane was marked with 7 mm × 7 mm squares. The membrane was then dipped in methanol for 10 s at room temperature and rinsed thoroughly with water before being laid on prewetted filter paper. A volume of 3 μL of serial dilutions of OTA-McAb in PBS were spotted onto the squares. Then the membrane was incubated at 37 °C for 2 h. After washing with PBST, the membrane was blocked by soaking into 3% skimmed milk in PBS for 1 h. After rinsing in PBST and drying, 7 mm squares were cut, dried, and kept at 4 °C until used. After the preparation of OTA-McAb spotted membrane, the assay was performed as follows: membrane squares were soaked in various concentrations of OTA standard or sample extract and containing the seleted phages (2.5 × 108 pfu/mL), reacted for 15 min at room temperature. After washing with PBST, the squares were incubated for 15 min by dipping into a 1/5000 dilution solution of HRP-conjugated anti-M13 antibody. Then the squares were washed and incubated with TMB substrate. Finally, the squares was rinsed with tap water, and the OTA concentration in the sample was analyzed visually. The color

intensities of OTA positive (different amount of OTA was mixed with phages) and sample extract were compared to the negative control (OTA was absent) visually. Validation of Phage-Based Immunoassays. Validation of the phage-based assays were performed by evaluating crossreactivity with other mycotoxins and by the determination of spiked samples and incurred samples. To evaluate cross-reactivity, mycotoxin standards (ochratoxin A, aflatoxin B1, aflatoxin M1, citrinin) at different concentrations (0, 0.001, 0.005, 0.025, 0.100, 0.500, 2.500, 10.000 ng/mL) were diluted in 5% (v/v) methanol/PBS applied to the chemiluminescent ELISA and mycotoxins at 0, 0.5, 1, 2.5, 5 ng/ mL in 10% (v/v) methanol/PBS were performed in the dipstick assay. To measure the recovery of OTA, instant coffee, corn, and rice samples were spiked with a known amount of OTA at final concentrations of 5, 10, 20, 50, and 100 μg/kg to OTA-negative samples. The sample extracts and dilution methods were described according to Wang et al.14 For instant coffee, 2 g of OTA-free instant coffee samples confirmed by HPLC were ultrasonically extracted for 20 min with 8 mL chloroform and centrifuged 5000g for 20 min. The chloroform layer (4 mL) was evaporated in a water bath and dissolved in 80 mL of 5% methanol/PBS for chemiluminescent ELISA analysis, dissolved in 10 mL of 10% methanol/PBS for dipstick assay, respectively. For corn and rice, 5 g of finely ground samples were mixed with 10 mL of 50% (v/v) methanol/water and ultrasonically extracted for 20 min. After centrifugation, the supernatant was diluted 5-fold with PBS for the dipstick assay, diluted 40-fold with 5% (v/v) methanol/PBS for the ELISA analysis, respectively. As to the analysis of incurred samples, cereals were prepared as above. While for the instant coffee, a cleanup step with a solid phase column was added prior to detection.

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RESULTS AND DISCUSSION Initial Panning in Random Peptide Library. During three rounds of selections, the phages from Ph.D.-12 with affnity to OTA-McAb were enriched from 4.7 × 105 pfu to 4.0 × 1010 pfu and eluted phages from Ph.D.-C7C were increased from 2.1 × 105 pfu to 9.0 × 109 pfu. After biopanning, 9 of 40 phage clones selected from the third round were identified to specifically bind to OTA-McAb competing with free OTA by phage ELISA (data not shown). Subsequently, phage single-stranded DNAs from the nine clones (Ph-1, Ph-5, Ph-6, Ph-7, Ph-12 and Ph-18 were selected from Ph.D.-12; Ph-4, Ph-10, and Ph-11 were selected from Ph.D.-C7C) were isolated, and their nucleotide sequences were determined. Among them, Ph-1 and Ph-6 have the identical sequence and so do the Ph-5 and Ph-12. As shown in Figure 2, four phages (Ph-1, Ph-7, Ph-11, and Ph-18) contains a motif FQLH, and the other three (Ph-4, Ph-5, and Ph-10) possessed FXLH sequence, where X is any amino acid residue. The seven isolates represented seven sequences were analyzed for OTAMcAb binding by competitive phage ELISA (Figure 2). Among them, Ph-7, with the motif of FQLH, exhibited the lowest IC50 of 0.50 ng/mL and a linear range of 0.26−0.93 ng/mL. While phage Ph-5, with the sequence of FXLH, showed the highest IC50 of 1.03 ng/mL, and the linear range was 0.41−2.56 ng/ mL. Consequently, we assume that FQLH are the key residues that are crucial for OTA-McAb binding, and it was seeded in the secondary library for evolution. 10306

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cysteine (C) residues were 6.7% and 0.3%, respectively, and did not match the expected frequencies (9.4% for arginine and 3.1% for cysteine). The reason might be the influence of R and C on phage assembly, infection, and secretion.32,34 Furthermore, serine (S) was overrepresented (12.3% observed vs 9.4% expected) in the constructed library, which coincide with that occurring in the commercial Ph.D. libraries. Besides, the observed frequency of each remaining residue was in conformity with that expected. These indicate that a high diversity is achieved in the second-generation peptide library. Second Panning in Second-Generation Peptide Library. After three rounds of selections from the secondgeneration library using competitive elution, phage indirect ELISA revealed 15 of 20 and 30 of 32 selected clones from the second and third round selections bound to OTA-McAb. Then the single-stranded DNA from 45 ELISA-positive phage clones were extracted and sequenced. Table 1 summarizes translated Table 1. Frequency of Amino Acids Occurred in SecondGeneration Peptide Library Phages Selected from the Second and Third Rounds of Biopanninga percentage of phages from selection round number position 1 2 3 4 5 6 7 8 9 10 11

Figure 2. Competitive inhibition curves of phages selected from (A) random peptide library and (B) second-generation peptide library. Conserved motif FQLH and GFQLH are in bold. Conservative substitutions in the motif are underlined and in bold. (C) Crossreactivity and sensitivity of the phage L12-311-based chemiluminescent ELISA. Error bars are standard deviations of the mean with n = 3.

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Characterization of Second-Generation Peptide Library. The secondary phage display peptide library contained the peptide sequences of AE-(X)3-FQLH-(X)3 was constructed according to the proposal designed by Beenhouwer et al. (Figure 1).27 “AE” residues lay near the signal peptide cleavage site because negatively charged amino acids (alanine (A) and glutamic acid (E)) should be the first choice in gram-negative signal peptidase cleavage sites.32 For cloning the peptide library, the ligation was optimized with 3:1, 5:1, and 10:1 ratios of cut duplex to cut vector. With the increase of the ratio, the amount of transformants was increased and reached the highest at 10:1. The total titer of the library was 1.2 × 108 pfu before amplification, and it is sufficient to encode all of the 6.4 × 107 (206) possible peptides theoretically. Following the construction of the second-generation library, the peptide sequences of 50 clones randomly picked up from the constructed library were determined. By comparing the observed frequency of each amino acid with that expected, the sequence diversity of the constructed library can be evaluated.33 The observed frequency of each amino acid = the number of times it occurred/300 residues (50 clones × 6 randomized residues) × 100%, while the expected frequency = the number of codons for that amino acid/32 codons ×100%. Of the 20 amino acids, the observed frequencies of the arginine (R) and

amino acid

2

3

alanine (A) glutamic acid (E) aspartic acid (D) D arginine (R) glycine (G) phenylalanine (F) glutamine (Q) leucine (L) histidine (H) serine (S) methionine (M) proline (P) R H

100 (15/15) 100 (15/15) 13 (2/15) 27 (4/15) 7 (1/15) 60 (9/15) 100 (15/15) 100 (15/15) 100 (15/15) 100 (15/15) 20 (3/15) 20 (3/15) 13 (2/15) 33 (5/15) 13 (2/15)

100 (30/30) 100 (30/30) 47 (14/30) 17 (5/30) 23 (7/30) 90 (27/30) 100 (30/30) 100 (30/30) 100 (30/30) 100 (30/30) 30 (9/30) 13 (4/30) 23 (7/30) 10 (3/30) 17 (5/30)

a

In total, 15 for the second round and 30 for the third round phage mimotopes were sequenced. The percentage of phage mimotope bearing the GFQLH sequence increased from 60% (9/15) to 90% (27/30) in phages obtained from the second and the third round, respectively.

sequences of these isolates. Analysis of the peptide sequences revealed a striking sequence convergence to a core motif (GFQLH). Of the round 2 isolates and round 3 isolates sequenced, isolates that possessed this core motif were increased from 60% to 90% (Table 1). The results suggesting that glycine is significant in conferring high specific binding to OTA-McAb except FQLH residues. Of the 45 ELISA-positive isolates, seven (L12-204, L12-206, L12-214, L12-311, L12-314, L12-317, and L12-330) showed lower IC50 values of 0.052−0.336 ng/mL than that phages selected from random peptide library by phage ELISA, which improved the sensitivity approximately 10-fold (Figure 2). For competitive inhibition curve established with phage L12-311, the IC50 was 0.052 ng/mL, and the linearity ranged from 0.008 to 0.330 ng/mL, revealing the highest sensitivity of the seven curves. So L12-311 was used in the following immunoassays. 10307

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Phage Chemiluminescent ELISA. The values of IC50, the maximum relative light unit (RLUmax), and the ratio of RLUmax to IC50 (RLUmax/IC50) were chosen as the parameters used for estimating the assay.10 After checkerboard assay, the reagents of OTA-McAb and phage with concentration of 2.5 μg/mL and 2.5 × 108 pfu/mL, respectively, were selected as the working conditions. The sensitivity and cross-reactivity of the method were determined as shown in Figure 2C, the limit of detection of the chemiluminescent ELISA, estimated from the mean (plus 2 standard deviations) of 10 blank samples, was 0.005 ng/mL, and IC50 was 0.04 ng/mL, linear range was 0.006−0.245 ng/ mL. Reproducibility and reliability of the ELISA assay were demonstrated by the coefficient variations (CVs) of 11.9% and 23.8% for intra-assay (evaluated repeatedly in different sets of wells of the same plate) and interassay (evaluated repeatedly in different plates in different days), respectively. According to the cross-reactivity test, no cross-reaction with aflatoxin B1, aflatoxin M1, and citrinin was found in chemiluminescent ELISA (Figure 2C). Methanol is the most commonly used in OTA standard and sample extraction. In order to evaluate the methanol effect, the concentration of 2.5%, 5%, 10%, and 20% methanol were examined, respectively, in the phage chemiluminescent ELISA (Figure 3A). The result showed a significant decrease of the maximum signal when a high concentration of methanol was used in the assay, which coincides exactly with the results reported previously.29 The lowest IC50 and the highest RLUmax/IC50 was observed at 5% methanol. It was reported that the reaction between antibody and antigen can be influenced by both pH and ionic strength.35,36 In addition, chemical compounds exist in sample extracts, such as solvent and salt might have effect on the pH and ionic strength. Thus these two parameters were generally investigated to evaluate the effect on an immunoassay.14,29 The influence of buffer ionic strength (from 5 to 50 mM) was investigated (Figure 3B). The obtained results indicated that the IC50 values were almost equal in 5 mM and 10 mM PBS, but the value of RLUmax/IC50 was higher when ionic strength was 10 mM. At the optimal ionic strength, the influence of pH was evaluated between 6.0 and 9.0 (Figure 3C). The maximum relative light unit decreased when the pH of assay buffer was lowered, and RLUmax/IC50 was the highest at pH 7.4. Taking account of the IC50 and RLUmax/IC50, the best performance was achieved at pH 7.4. Thus, 5% methanol in 10 mM PBS, pH 7.4 were selected as a working solution for the assay. Phage Dipstick Assay. Besides the chemiluminescent ELISA, phage L12-311 was also applied in the noninstrumental dipstick assay. In the inhibition assay for mycotoxins, the sensitivity increases with high dilutions of antibody and antigen conjugate.31 For the purpose of determining the appropriate dilution of the immunoassay reagents, a checkerboard assay was conducted by using different dilutions of OTA-McAb and phage (Figure 4). The assay incubated with 40 μg/mL OTAMcAb and 1/8000 dilution of phages was chosen to be the minimum concentration of reagents for a clear and high enough intensity of dot (Figure 4A). Thus, 40 μg/mL OTA-McAb and 8000-fold dilution of phage (2.5 × 108 pfu/mL) was used in further experiments. Because methanol used for preparation of OTA standard and extraction of OTA from food samples generally affects antigen−antibody interaction and it may reactivate the PVDF membrane after pretreatment, the appropriate concentration

Figure 3. Effects of (A) methanol, (B) ionic strength, and (C) pH on the performance of the phage-based chemiluminescent ELISA. Each value represents the mean of triplicate assays.

Figure 4. Dipstick assay for OTA. (A) OTA-McAb (ranging from 20 to 160 μg/mL) spotted onto PVDF membranes were incubated with different dilutions of phage particles (ranging from 1/2 000 to 1/16 000). (B) Dipstick assay set with various concentrations of OTAMcAb (20−160 μg/mL) and using 1/8 000 dilution of phage (2.5 × 108 pfu/mL) for detection. The concentration of OTA ranged from 0 to 5 ng/mL as shown in the right panel.

(10%, 20%, and 40%) of methanol was examined. With an increase of methanol concentration, high backgound color was observed in unspotted areas. Furthermore, the sensitivity was decreased at 40% methanol/PBS. The effect was the lowest at 10% methanol/PBS and indicating that methanol interference was not significant. 10308

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Table 2. Recoveries of OTA Added to Corn, Rice, and Instant Coffee Samples in Determinations Performed by Phage-Based Chemiluminescent ELISA, Conventional ELISA, and Phage-Based Dipstick Assay phage-based chemiluminescent ELISA (n = 3) matrix corn

rice

instant coffee

a

OTA added (μg/kg) 5 10 20 50 100 5 10 20 50 100 5 10 20 50 100

OTA recovered (μg/kg) 6.2 11.1 17.3 40.0 72.0 4.2 9.3 19.4 61.8 86.0 4.3 8.0 13.2 26.1 42.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.9 2.5 2.7 17.3 1.0 1.8 1.5 7.7 8.5 0.6 1.3 2.0 7.6 12.8

recovery (%) 124.0 111.0 86.5 80.0 72.0 84.0 93.0 97.0 123.6 86.0 86.0 80.0 66.0 52.2 42.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

conventional ELISA (n = 3) OTA recovered (μg/kg)

16.0 9.0 12.5 5.4 17.3 20 18.0 7.5 15.4 8.5 12.0 13.0 10.0 15.2 12.8

5.7 12.4 23.0 48.2 70.2 4.6 8.9 19.0 58.8 85.4 3.9 7.6 9.8 31.3 44.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.6 0.3 0.8 2.8 1.0 0.5 0.7 0.6 1.7 6.3 0.9 0.8 0.6 2.5 3.6

recovery (%)

phage-based dipstick assay (n = 3)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

−a− − +b ±c ± +++ +++ +++ −−− ±− ± ++± +++ +++ −−− −−− ±− − +++ +++

114.0 124.0 115.0 96.4 70.2 92.0 89.0 95.0 117.6 85.4 78.0 76.0 49.0 62.6 44.0

12.0 3.0 4.0 5.6 1.0 10.0 7.0 3.0 3.4 6.3 18.0 8.0 3.0 5.0 3.6

Negative, an obvious dot was observed. bPositive, no dot was observed. cNegative/positive, an unclear dot was observed.

also effective to obtain a good recovery from green coffee beans.39 A total of 40 samples of foodstuffs and feedstuffs obtained from the Chinese market were analyzed using phage-based chemiluminescent ELISA, commercial ELISA kit, and phagebased dipstick assay, respectively. Among the 40 samples, 4 (C3, C4, C6, C7) of 10 corn samples, 7 (B1, B2, B3, B5, B6, B8, B10) of 10 barley samples, 6 (IC4, IC5, IC6, IC7, IC9, IC10) of 10 instant coffee samples, and 1 (F5) of 5 feedstuffs were OTA positive and 5 rice samples were all OTA negative detected by the chemiluminescent ELISA (Table 3). The limits of detection (or cutoff level) of the three methods were 0.4, 5, and 10 μg/kg, respectively. A total of 18 out of the 40 samples tested positive for OTA by the chemiluminescent ELISA as shown in Table 3. In addition, OTA was detected in 7 samples (F5, B2, B3, IC5, IC6, IC7, and IC10) by the ELISA kit and two samples (F5 and B2) by the dipstick assay. Much more samples were determined by the phage-based chemiluminescent ELISA to be OTA positive because of the higher sensitivity compared to the ELISA kit and dipstick assay. The results obtained from the three methods were in agreement with each other.

To estimate the cutoff level (the minimum concentration of mycotoxin capable of achieving 100% inhibition and thus no spot was observed) of the dipstick assay, OTA was analyzed at different concentrations (Figure 4B). With an increase of OTA concentration, the intensity of the spot color decreased. The spot was invisible when the OTA concentration increased to 1 ng/mL. Validation Studies. The validations were performed by analyzing the spiked samples using chemiluminescent ELISA, dipstick assay, and a conventional indirect competitive ELISA, respectively. As presented in Table 2, cereals spiked with 5− 100 μg/kg OTA exhibited recoveries of 72.0−124.0% for corn and 84.0−123.6% for rice by the phage chemiluminescent ELISA, while by conventional ELISA the values were 70.2− 124.0% for corn and 85.4−117.6% for rice. As for the dipstick assay, clear dots were observed for corn and rice spiked with 5 μg/kg OTA but unclear dots or no dots were obtained from corn and rice spiked with 10, 20, 50, and 100 μg/kg OTA. These results demonstrated that adequate recovery and efficiency from cereals were achieved with phage-based OTA mimotope. However, instant coffee had low OTA recoveries analyzed by both phage chemiluminescent ELISA and conventional ELISA (Table 2). By phage chemiluminescent ELISA, OTA levels ranging of 5−100 μg/kg resulted in recoveries of 42.9−86.0% and conventional ELISA showed similar data (44.0−78.0%). The same pattern was observed by dipstick assay (Table 2). The low recovery applies to many immunoassays for OTA analysis from coffee samples. The OTA recovery determined by ELISA was 74−76% for coffee with spiked OTA ranging from 2.5 to 10 μg/kg.14 Unsatisfactory recoveries from roasted coffee and instant coffee were also reported by Fujii et al.37 The problem was probably due to the pigment or other complex components of coffee which interfered with the antigen− antibody binding.14 In order to get a high recovery from coffeebased samples, it seems that it is essential to add a cleanup step before analysis. Better recoveries (92.8% for green coffee by liquid chromatography) were obtained by purifying with an immunoaffinity column cleanup.38 A solid-phase extraction was

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CONCLUSIONS In this study, OTA mimotopes were initially selected from random peptide libraries and used to confirm a motif sequence. On the basis of the motif, a second-generation peptide library was then constructed and various affinities of mimotopes were obtained. The sensitivities of the ELISA set up with the mimotopes selected from the second-generation peptide library improved significantly. An OTA mimotope from the constructed library was selected and applied to chemiluminescent ELISA and dipstick assay for the detection of OTA. The IC50 value of the chemiluminescent ELISA was 0.04 ng/mL, and the dipstick assay has a visual cutoff level of 1 ng/mL. Results obtained from phage-based chemiluminescent ELISA, conventional ELISA, and phage-based dipstick assay were also in good agreement with each other. To our best knowledge, it has not been reported that the affinity of ochratoxin A mimotope can be developed by selecting second-generation 10309

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Table 3. Analysis of Incurred Samples by Phage-Based Chemiluminescent ELISA, Commercial ELISA Kit, and Phage-Based Dipstick Assaya

sample feed corn

barley

instant coffee

number

phage-based chemiluminescent ELISA (μg/kg, n = 3)

commercial ELISA kit (μg/kg, n = 3)

phage-based dipstick assay (μg/kg, n = 3)

F5 C3 C4 C6 C7 B1 B2 B3 B5 B6 B8 B10 IC4

17.21 0.65 2.40 1.28 1.30 4.12 11.73 5.44 1.20 3.73 2.46 0.70 3.21

± ± ± ± ± ± ± ± ± ± ± ± ±

1.23 0.07 0.31 0.09 0.14 0.27 0.43 0.15 0.16 0.25 0.13 0.06 0.27

16.07 ± 1.77 NDc NDc NDc NDc NDc 13.31 ± 0.25 6.08 ± 0.32 NDc NDc NDc NDc NDc

+b + + −d− − −−− −−− −−− −−− +++ −−− −−− −−− −−− −−− −−−

IC5 IC6 IC7 IC9 IC10

4.87 6.13 6.40 3.70 4.52

± ± ± ± ±

0.58 0.43 0.18 0.23 0.09

7.53 ± 8.24 ± 5.43 ± NDc 6.24 ±

− − − − −

0.44 0.32 0.08 0.31

− − − − −

− − − − −

a

The limits of detection (or cut-off level) of the phage-based chemiluminescent ELISA, commercial ELISA kit, and phage-based dipstick assay were 0.4, 5, and 10 μg/kg, respectively. bPositive, no dot was observed. cNot detectable. dNegative, an obvious dot was observed.

peptide library. The result of this work provides a model significance for other mycotoxins and toxic small molecules in the application of mimotopes.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-791-88305177-8105. Fax: +86-791-88333708. Email: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from National Basic Research Program of China (Grant 2013CB127804), the National Natural Science Funds of China (Grants NSFC31360386, NSFC-31201360, and NSFC-31171696), National Key Technology R & D Program of “12th Five-Year Plan” (Grant 2012BAK17B02), 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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