Phage-Mediated Immuno-PCR for Ultrasensitive Detection of Cry1Ac

Sep 29, 2016 - The widespread use of Cry proteins in transgenic plants for insect control has raised concerns about the environment and food safety in...
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Phage-Mediated Immuno-PCR for Ultrasensitive Detection of Cry1Ac Protein Based on Nanobody Yuanyuan Liu,∥ Dongjian Jiang,∥ Xin Lu, Wei Wang, Yang Xu, and Qinghua He* State Key Laboratory of Food Science and Technology, Sino−German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China

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S Supporting Information *

ABSTRACT: The widespread use of Cry proteins in transgenic plants for insect control has raised concerns about the environment and food safety in the public. An effective detection method for introduced Cry proteins is of significance for environmental risk assessment and product quality control. This paper describes a novel phage mediated immuno-PCR (iPCR) for the ultrasensitive determination of Cry proteins based on nanobodies. Three nanobodies against Cry1Ac protein were obtained from a naı̈ve phage displayed nanobody library without animal immunization process and were applied to the iPCR assay for Cry1Ac. The phage-mediated iPCR for Cry1Ac based on nanobodies showed a dynamic range of 0.001−100 ng/mL and a limit detection of 0.1 pg/mL. Specific measurement of this established method was performed by testing cross-reativity of other Cry1Ac analogues, and the result showed negligible cross-reactivity with other test Cry proteins (Cry1Ab, Cry1F, Cry3B). Furthermore, the phage-mediated iPCR based on nanobody should be easily applicable to the detection of many other Cry proteins. KEYWORDS: Cry1Ac protein, nanobody, ommuno-PCR, immunoassay

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INTRODUCTION Insecticidal formulations containing crystalline (Cry) proteins, which were discovered from Bacillus thuringiensis (Bt) species, have been used to control pest insects for decades.1 Currently, the use of transgenic plants (including tobacco, cotton, rice, potato, and maize plants) producing Cry proteins has rapidly increased. For example, as of 2015, 81% of maize and 84% of cotton planted in the United States expressed one or more insecticidal traits for protection against a diversity of insect pests.1,2 However, the widespread use of Cry proteins in transgenic plants for insect control has raised concerns about the problem of gene flow, insect resistance, and allergenicity caused by Cry proteins.3−5 As such, a rapid, sensitive, and selective analytical method for introduced genes or their expressed proteins is important for environmental and food safety.6,7 To date, various kinds of analytical methods have been developed to detect the presence of Cry proteins (or their genes) in the food and supply chain. As to DNA-based methods, various types of polymerase chain reaction (PCR) assays have been developed for detecting introduced genes in transgenic plants,8−10 whereas protein-based immunoassay methods include enzyme-linked immunosorbent assay (ELISA),1,6,11 immunochromatographic strip test,12 immunosensors,13 and Western blotting.1 The protein-based immunoassay that uses Cry protein-specific antibody is highly specific and is suitable for high sample throughput, but lacks the promising sensitivity of the PCR assay.14 In contrast, immuno-PCR (iPCR) offers a more sensitive detection method than existing protein-based immunoassays.15 iPCR combines the specificity of antibody-based immunoassay with the sensitivity of PCR assay.16 To date, several iPCR assays for Cry proteins (Cry1Ac and Cry1Ab) have been developed.17,18 © 2016 American Chemical Society

Generally, linking detection monoclonal antibody (mAb) to oligonuleotides that can be amplified by PCR with appropriate primers is a necessary and important step to establish an iPCR assay.19 Although the chemical synthetic method for mAb−DNA conjugates is mature, it is time-consuming and complicated to perform. To avoid these problems, a phage display mediated iPCR assay was reported with the development of phage displayed single-chain variable fragments (ScFv) technology.20 For the phage displayed ScFv, ScFv fragments that can bind to a specific target were displayed on the surface of phage particles, and genes coding ScFv were fused into the phage DNA, which means the phage displayed ScFv can simultaneously serve as both detection antibody and PCR DNA template.20 However, ScFv lacks the affinity of the parent mAb, and preparing ScFv to a specific target is time-consuming and labor-intensive.21 In this study, we describe a novel phage-mediated iPCR assay for the ultrasensitive determination of Cry proteins based on nanobody. A nanobody, also called a single-domain antibody, is an antibody fragment consisting of a single monomeric variable antibody domain.22 Compared to other antibody fragments, a nanobody has many advantages including excellent solubility, thermal stability, and ease of expression.23,24 Furthermore, researchers can conveniently obtain nanobodies against a specific target from a naı̈ve phage displayed nanobody libarary without animal immunization process. Here, Cry1Ac was used as a model of Cry proteins, and nanobodies against Cry1Ac protein were obtained from a naı̈ve phage displayed nanobody library without an animal immunization process. On this basis, a phage-mediated Received: Revised: Accepted: Published: 7882

July 3, 2016 September 25, 2016 September 29, 2016 September 29, 2016 DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

Article

Journal of Agricultural and Food Chemistry

Figure 1. Schematic presentation of the biopanning, expression of nanobody that binds to Cry1Ac protein, and its application in iPCR assay. Expression and Purification of Soluble Anti-Cry1Ac Nanobody. Nanobody genes from selected positive phage clones were isolated by using a plasmid extraction kit (DP106,Tiangen, Beijing). After the size and purity of extracted nanobody genes had been verified by 0.8% agarose gel electrophoresis, the plasmid DNAs were recloned into expression vector pET-25(+) using restriction enzymes NotI and NocI, and then plasmid constructs were transformed into E. coli Rosetta competent cells by electroporation and cultured in LB medium (containing 100 μg/mL ampicillin and chloramphenicol). Individual colonies from LB plates were picked up and used for the expression of periplasmic soluble nanobodies by adding 0.2 mM IPTG into culture and incubation was continued for 6 h at 32 °C. After the cells had been collected and ultrasonically broken, periplasmic extract was loaded in a Ni-chelating affinity chromatography column for purifying. The bound soluble nanobodies with 6×His tag were eluted with a 200 mM imidazole buffer.27 Then, nanobody eluate was dialyzed against 0.01 M PBS, and 50 μL of dialyzed fluid was pipetted to SDS-PAGE analysis to verify the prepared soluble nanobody’s size and purity. Sandwich ELISA for Cry1Ac-Based Nanobody. The soluble antiCry1Ac nanobody and phage displayed nanobody against Cry1Ac were used as capture and detection antibody, respectively, to develop a sandwich ELISA for Cry1Ac. For the purpose of evaluating the desired performance of nanobody-based sandwich ELISA for Cry1Ac, a checkerboard assay was performed in advance to optimize the working concentration of capture and detection antibody as described by Shan et al.6 The procedure of nanobody-based sandwich ELISA for Cry1Ac was as follows: First, 100 μL of soluble anti-Cry1Ac nanobody was coated in microplate wells at 4 °C for 12 h, and then wells were blocked with 5% skim milk (diluted in 0.01 M PBS) for 2 h at 37 °C. Second, after 10 washings with PBST, 50 μL of Cry1Ac standard at various concentrations or samples and 50 μL of phage displayed nanobody against Cry1Ac were put into respective wells for 30 min of incubation at 37 °C. Third, nonbinding nanobody was poured off and discarded, and wells were washed 10 times with PBST; 100 μL of a 1:5000 dilution of anti-M13 phage coat protein antibody conjugated with HRP was incubated in the wells at 37 °C for 30 min. Finally, 100 μL of TMB substrate was pipetted into each well and incubated at 37 °C for 10 min.

iPCR assay based on nanobodies was established to detect Cry1Ac protein.

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MATERIALS AND METHODS

Chemicals and Reagents. Bt toxic proteins Cry1Ac, Cry1Ab, Cry1F, and Cry3B were purchased from Youlong Co., Ltd. (Shanghai,China). Ni-affinity chromatography, anti-M13 antibody coupled to peroxidase, and anti-6×His antibody coupled to peroxidase were purchased from GE Healthcare (Piscataway, NJ, USA). Isopropylthio-βD-galactoside (IPTG), bovine serum albumin (BSA), ovalbumin (OVA), and TMB chromogenic substrate were obtained from SigmaAldrich Co. (St. Louis, MO, USA). Restriction enzymes NotI and NcoI, T4 DNA ligase, and TaqDNA polymerase were purchased from Takara Co. (Dalian, China). pET25b (+) vector, Escherichia coli Rosetta, was prepared in our laboratory. Selection of Phage Displayed Nanobody against Cry1Ac. Here, a naı̈ve nanobody phage displayed library24 was used for biopanning of nanobodies against Cry1Ac protein. The biopanning procedures were described by previous work.25 A schematic diagram for biopanning procedures is present in Figure 1. Briefly, for the first round of biopanning, 100 μL of Cry1Ac (50 μg/mL, diluted in 0.01 M PBS) was put into microplate wells and incubated at 4 °C for 12 h. To block the uncoated area, 300 μL of 3% BSA−PBS was added into wells and incubated for 60 min at 37 °C. After 10 washings by 0.1% PBST (Tween 20, v/v %), 100 μL of naı̈ve phage display nanobody library (2.0 × 1011 pfu) was added into wells and incubated at 37 °C for 60 min with gentle shaking. Nonbinding phages were discarded, and wells were washed 10 times by 0.1 % PBST buffer. Bound phages were eluted using 0.2 M glycine-HCl (pH 2.2, 100 μL), and the elution was rocked for 10 min at 37 °C with gentle agitation. The eluate was pipetted into a 1.5 mL centrifuge tube and neutralezed with 1 M Tris-HCl (pH 9.1, 15 μL) immediately. For next three rounds, the added phage amounts remained the same (2.0 × 1011 pfu), whereas Cry1Ac coating concentration was reduced to 25, 15, and 10 μg/mL, respectively. After biopanning procedures, phage-ELISA was performed to identify positive phage that can bind to Cry1Ac, and then coding DNA of positive phage was sequenced according to the described previous work.22,24,26 7883

DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

Article

Journal of Agricultural and Food Chemistry

Figure 2. (A) Number of phage output in each round of biopanning; (B) phage ELISA for identifying the positive phage clones binding to Cry1Ac protein; (C) three kinds of different amino acid sequences of phage displayed nanobodies against Cry1Ac (from top to bottom: P10, P24, and P72, respectively). After 50 μL of 2 M H2SO4 stop solution had been added into each well, the microplate wells were read using a microplate reader at 450 nm. Phage-Mediated iPCR for Cry1Ac. Anti-Cry1Ac soluble nanobody (0.5 μg/mL, 100 μL/well) was coated in glutaraldehyde-modified

wells and blocked as described for sandwich ELISA procedures. Next, equal parts phage displayed nanobody and Cry1Ac standard (50 μL) were mixed, coated into wells, and incubated at 37 °C for 30 min. Then, after 10 washings with 0.1% PBST, the bound phage displayed 7884

DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

Article

Journal of Agricultural and Food Chemistry

Figure 3. Reaction curves of sandwich ELISA for Cry1Ac based on different pairwise nanobodies against Cry1Ac. Each value is the average of three experimental replicates ± standard deviation.

Figure 4. Standard curve of sandwich ELISA for Cry1Ac based on N24/P72 paired nanbodies (capture and detection antibodies are N24 and P72, respectively). Each value is the average of three experimental replicates ± standard deviation. Assessment of the Phage-Mediated iPCR by Spiked Samples. To identify this established method’s accuracy, various concentrations of Cry1Ac protein were spiked in corn and wheat samples. One gram of each sample was prepared by spiking with a known concentration level of Cry1Ac protein (0.1, 1, 10, or 100 ng/mL), and the mixture were extracted with 1 mL of extraction solution (0.1 M PBS, containing 0.2% BSA and 0.1% Tween-20)6 for 10 min at 37 °C. After centrifugation, supernatant was directly applied to phage-mediated iPCR assay.

nanobody was eluted using 0.2 M glycine-HCl (pH 2.2) with gentle rocking for 10 min. The eluted phages were immediately neutralized with 15 μL of 1 M Tris-HCl (pH 9.1) and directly worked as DNA template in iPCR. All of the phage-mediated iPCRs were carried out by 7900HT PCR machine (ThermoFisher). A single PCR reaction requires SYBR* PremixEx TaqII (10 μL), primers (forward, 5′-ACA CGG TGT TTC TGC AAA TGA-3′, 10 mM in 1 μL; reverse, 5′-GCG AAT AAT ACC AGC TAC CAG-3′, 10 mM in 1 μL), phages (109 pfu/mL in 5 μL), and distilled water. The step program for PCR thermal cycle was as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and then 72 °C for 30 s. No DNA template well was used as negative control. To evaluate cross-reactivity, nontarget Bt toxic proteins (Cry1Ab, Cry1F, Cry3B) in assay buffer were applied to nanobody-based iPCR assay, respectively.

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RESULTS AND DISCUSSION

Selection of Phage-Displayed Nanobodies against Cry1Ac. Phages with affinity to Cry1Ac protein were enriched after biopanning elution (Figure 2A). Competitive phage ELISAs were carried out to identify 72 randomly picked phage clones (derived from the eluates of a fourth round of panning elution). 7885

DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

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Journal of Agricultural and Food Chemistry

Figure 5. Effect of (A) pH and (B) ionic strength on the performance of N24/P72 paired nanobody-based sandwich ELISA for Cry1Ac; (C) thermal stability analysis of nanobody N24 by ELISA after treatment at 37, 45, 55, 65, 75, and 85 °C in a water bath for 10 min, respectively. The activity of N24 incubated at 37 °C was regarded as 100%.

Among these 72 clones, 22 showed different degrees of binding to Cry1Ac (Figure 2B). DNA sequencing results showed that these 22 phage clones were 3 virtual clones (P10, P24, and P72; Figure 2C). To obtain phages that can bind to a specific target from a naı̈ve phage displayed libarary, we performed a stringent panning

strategy as follows: First, the concentration of coated Cry1Ac was decreased stepwise (50, 25, 15, and 10 μg/mL) in four rounds of biopanning. Second, it was reported that the concentration of Tween in washing buffer is important, affecting the titer of eluate.28 In this study, the enrichment of phage output was observed by gradually increasing the concentration of Tween 7886

DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

Article

Journal of Agricultural and Food Chemistry

Figure 6. Quantitative detection of standard Cry1Ac solution by phage-displayed nanobody mediated iPCR: (A) real-time PCR amplification curves (curves represent serial dilution of Cry1Ac from 0.001 to 100 ng/mL); (B) standard curve of nanobody (N24/P72 pair) mediated iPCR for Cry1Ac. Each value is the average of three experimental replicates ± standard deviation.

minimized, and these selected phage clones have low affinity with BSA and OVA. Expression of Soluble Nanobody against Cry1Ac. To express soluble nanobodies, the VHH genes of Cry1Ac binders (P10, P24, and P72) were subcloned into vector pET25b(+) to construct expression plasmid for nanobody. After DNA sequencing, the results showed that all three constructed plasmids exhibited expected DNA sequences (data not shown). To get amounts of soluble nanobodies, culture parameters including the concentrations of inducer IPTG, inoculation time, and temperature were evaluated. After an orthogonal experiment, inducer IPTG with a concentration of 0.2 mM and cultured at 32 °C for 6 h was selected as the nanobody expression condition. Under this culture condition, the yield of soluble nanobodies ranged from 25 to 35 mg/L. The soluble nanobody can be conveniently purified by Ni columns because it contains a 6×His tag, and SDS-PAGE analysis results showed purified soluble nanobody with >95% purity (Figure S-

Table 1. Recoveries of Cry1Ac from Spiked Samples by Nanobody-Based iPCR Assay added Cry1Ac concentration (ng/mL)

found Cry1Ac concentration (ng/mL)

recovery (%)

corn

0.1 1 10 100

0.116 ± 0.01 1.02 ± 0.084 11.40 ± 1.56 78.81 ± 4.83

116.0 102.0 114.0 78.81

wheat

0.1 1 10 100

0.075 ± 0.009 0.79 ± 0.074 11.40 ± 1.04 85.28 ± 8.04

75.0 79.0 114.0 85.28

sample

(0.1, 0.25, 0.35, and 5%) in four rounds of panning. Third, the BSA−PBS and OVA−PBS blocking buffers were used interchangeably in each round of panning to remove phage nanobody clones binding the nontarget protein from the blocking buffer, nonspecific phage nanobody clones were 7887

DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

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Journal of Agricultural and Food Chemistry

demonstrated that adequate recovery was achieved with phagemediated iPCR for Cry1Ac based on nanobody.

1). The purified soluble nanobodies, corresponding to P10, P24, and P72, were named N10, N24, and N72, respectively. Nanobody-Based ELISA for Cry1Ac. Soluble nanobody was paired with phage displayed nanobody against Cry1Ac to develop a sandwich ELISA for Cry1Ac. Figure 3 shows that N10 can form pairs with P24 and P72 with SC50 values (concentration values causing 50% signal saturation) of 27.17 ± 1.76 and 21.87 ± 2.29 ng/mL, respectively. Soluble nanobody N72 can form valuable pairs with P24 and P72, but did not form a valuable pair with P10. Curiously, soluble nanobody N24 can form valuable pairs with any of the other antibodies (including the phage displayed nanobody P24, which has the same nanobody sequence as N24), and the SC50 value of the N24/P24 paired assay was 8.10 ± 0.41 ng/mL. On the basis of the overall results of the pairwise screening, we chose to optimize the N24/P72 paired assay. Under the optimal conditions, the soluble nanobody N24 and phage displayed nanobody P72 with concentrations of 1.0 μg/mL and 2.0 × 1010 (pfu/mL) were selected as working conditions. The results of Figure 4 show that the N24/P72 paired assay has a dynamic range of about 4 logs with the limit of detection (LOD), defined as 3 times the standard deviation of the blank, of 0.08 ng/mL. It was reported that ionic strenth and pH value can affect the performance of the immunoassay.24 After the nanobody-based sandwich ELISA assay had been performed at various values of ionic strength (from 5 to 40 mM) and pH (from 5.7 to 9.0) buffer, the results showed that nanobody has promising ionic strength and pH tolerance ability (Figure 5A,B). Furthermore, the thermal stability of the nanobody was evaluated by incubating nanobody (N24) for 10 min at 37, 45, 55, 65, 75, and 85 °C in a water bath and applied to Cry1Ac-coated plates for performance of the ELISA (the activity of N24 incubated at 37 °C was regarded as 100%). As shown in Figure 5C, the activity of nanobody (N24) was maintained at 90% after incubation for 10 min at 55 °C. Even after incubation at 85 °C, N24 activity was maintained at nearly 63%. Nanobody-Based Quantitative iPCR for Cry1Ac. The procedure of nanobody-mediated iPCR was similar to sandwich ELISA. Initially, a series of standard Cry1Ac solutions with different concentrations were prepared and detected in triplicate by phage-mediated real-time PCR. As Figure 6A shows, the Ct (cycle threshold number) value is gradually increased with the decrease of Cry1Ac protein concentration (from 0.001 to 100 ng/mL). Then, a standard curve of phage-mediated iPCR was constructed by plotting Cry1Ac concentration against the mean Ct value; the correlation coefficient of the standard curve was 0.996, which confirmed the accuracy of the quantification (Figure 6B). The phage-mediated iPCR for Cry1Ac based on nanobodies showed a dynamic range of 0.001−100 ng/mL and a limit detection of 0.1 pg/mL, which was more sensitive than an analogous ELISA. Specific measurement of this method was performed by comparing SC50 with those of other Cry1Ac analogues (including Cry1Ab, Cry1F, and Cry3B). Crossreactivity (CR) was calculated as CR% = SC50(Cry1Ac)/ SC50(tested analogues) × 100%. The result showed that negligible CR was observed with the double nanobody mediated iPCR for Cry1Ac (Figure S-2). Furthermore, the method accuracy of nanobody-based iPCR was evaluated by spiking Cry1Ac negative samples (which were previously tested by Cry1Ac test kit) with different concentration of Cry1Ac (from 0.1 to 100 ng/mL). As Table 1 shows, the average recoveries ranged from 75 to 116%. These results

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Puyblications website at DOI: 10.1021/acs.jafc.6b02978 Figures S-1 and S2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(Q.H.) Phone: +86-791-8830 5177. E-mail: heqinghua@ncu. edu.cn. Author Contributions

∥ Y.L. and D.J. contributed equally to this work and should be considered co-first authors

Funding

This work was supported financially by grants from the National Natural Science Funds (Grants NSFC-31360386, NSFC31671924, and NSFC-31171696), the Jiangxi Province Key Technology R&D Program (Grant 20141BBG70090), a major program of the Natural Science Foundation of Jiangxi, China (Grant 20143ACB21008), and a grant of the Education Department of Jiangxi Province (Grant GJJ150007). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Vurtice, V. C.; Hellmich, R. L.; Coats, J. R. A review of Cry protein detection with enzyme-linked immunosorbent assays. J. Agric. Food Chem. 2016, 64, 2175−2189. (2) Acreage; U.S. Department of Agriculture, National Agricultural Statistics Service: Washingtion, DC, USA, 2015. (3) Zhao, Z.; Chen, Y.; Xu, W.; Ma, M. Surface plasmon resonance detection of transgenic Cry1Ac cotton (Gossypium spp.). J. Agric. Food Chem. 2013, 61, 2964−2969. (4) Snow, A. A.; Andow, D. A.; Gepts, P.; Hallerman, E. M.; Power, A.; Tiedje, J. M.; Wolfenbarger, L. L. Genetically engineered organisms and the enviroment: current status and recommendations. Ecol. Appl. 2005, 15, 377−404. (5) Lu, Y.; Wu, K.; Jiang, Y.; Xia, B.; Li, P.; Feng, H.; Wyckhuys, K. A.; Guo, Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adopation of Bt cotton in China. Science 2010, 328, 1151−1154. (6) Shan, G.; Embrey, S. K.; Schafer, B. W. A highly specific enzymelinked immunosorbent assay for the detection of Cry1Ac insecticidal Crystal protein in transgenic widestrike cotton. J. Agric. Food Chem. 2007, 55, 5974−5979. (7) Stave, J. W. Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations. J. AOAC Int. 2002, 85, 780−786. (8) Randhawa, G. J.; Chhabra, R.; Singh, M. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screnning of genetically modified crops. J. Agric. Food Chem. 2009, 57, 5167−5172. (9) Dinon, A. Z.; Prins, T. W.; Van Dijk, J. P.; Arisi, A. C.; Scholtens, I. M.; Kok, E. J. Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modificed organisms. Anal. Bioanal. Chem. 2011, 400, 1433−1442. (10) Guan, X.; Guo, J.; Shen, P.; Yang, L.; Zhang, D. Visual and rapid detection of two genetically modified soybean events using loopmediated isothermal amplification method. Food Anal. Methods 2010, 3, 313−320. (11) Nguyen, H. T.; Jehle, J. A. Quantitative analysis of the seaonal and tissue-specific expression of Cry1Ab in transgenic maize Mon810. J. Plant Dis. Prot. 2007, 114, 82−87.

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DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889

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Journal of Agricultural and Food Chemistry (12) Santos, V. O.; Pelegrini, P. B.; Mulinari, F.; Moura, R. S.; Cardoso, L. P. V.; Buhrer-Sekula, S.; Miller, R. N.G.; Pinto, E. R. C.; Grossi-de-Sa, M. F. A novel immunochromatographic strip test for rapid detection of Cry1Ac and Cry8Ka5 proteins in genetically modified crops. Anal. Methods 2015, 7, 9331−9339. (13) Volpe, G.; Ammid, N. H.; Moscone, D.; Occhigrossi, L.; Palleschi, G. Development of an immunomagnetic electrochemical sensor for detection of BT-CRY1AB/CRY1AC proteins in genetically modificed corn samples. Anal. Lett. 2006, 39, 1599−1609. (14) Randhawa, G.; Singh, M.; Sood, P. DNA-based methods for detection of genetically modified events in food and supply chain. Curr. Sci. 2016, 110, 1000−1009. (15) Niemeyer, C. M.; Adler, M.; Wacker, R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol. 2005, 23, 208−216. (16) Adler, M.; Wacker, R.; Niemeyer, C. M. A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins. Biochem. Biophys. Res. Commun. 2003, 308, 240−250. (17) Kumar, R. A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein. Eur. Food Res. Technol. 2012, 234, 101−108. (18) Allen, R. C.; Rogelj, S.; Cordova, S. E.; Kieft, T. L. An immunoPCR method for detecting Bacillus thuringiensis Cry1Ac toxin. J. Immunol. Methods 2006, 308, 109−115. (19) Malou, N.; Raoult, D. Immuno-PCR: a promising ultrasensitive diagnostic method to detect antigens and antibodies. Trends Microbiol. 2011, 19, 295−302. (20) Guo, Y.; Zhou, Y.; Zhang, X.; Zhang, Z.; Qiao, Y.; Bi, L.; Wen, J.; Liang, M.; Zhang, J. Phage display mediated immuno-PCR. Nucleic Acids Res. 2006, 34, e62. (21) Pini, A.; Bracci, L. Phage display of antibody fragments. Curr. Protein Pept. Sci. 2000, 1, 155−169. (22) Chen, J.; He, Q.; Xu, Y.; Fu, J.; Li, Y.; Tu, Z.; Wang, D.; Shu, M.; Qiu, Y.; Yang, H.; Liu, Y. Nanobody mediated immunoassay for ultrasensitive detection of cancer biomarker α-fetoprotein. Talanta 2016, 147, 523−530. (23) Kierny, M. R.; Cunningham, T. D.; Kay, B. K. Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms. Nano Rev. 2012, 3, 17240. (24) Qiu, Y.; He, Q.; Xu, Y.; Bhunia, A. K.; Tu, Z.; Chen, B. Deoxynivalenol-mimic nanobody isolated from a naive phage display nanobody library and its application in immunoassay. Anal. Chim. Acta 2015, 887, 201−208. (25) Kim, H. J.; McCoy, M. R.; Majkova, Z.; Dechant, J. E.; Gee, S. J.; Tabares-da Rosa, S.; Gonzalez-Sapienza, G. G.; Hammock, B. D. Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal. Chem. 2012, 84, 1165− 1171. (26) Xu, Y.; Chen, B.; He, Q.; Qiu, Y.; Liu, X.; He, Z.; Xiong, Z. New approach for development of sensitive and enviromentally friendly immunoassay for mycotoxin fumonisin B1 based on using peptide-MBP fusion protein as substitute for coating antigen. Anal. Chem. 2014, 86, 8433−8440. (27) Beuerle, T.; Pichersky, E. Enzymatic synthesis and purification of aromatic coenzyme A esters. Anal. Biochem. 2002, 302, 305−312. (28) He, Q.; Xu, Y.; Huang, Y.; Liu, R.; Huang, Z.; Li, Y. Phagedisplayed peptides that mimic zearalenone and its application in immunoassay. Food Chem. 2011, 126, 1312−1315.

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DOI: 10.1021/acs.jafc.6b02978 J. Agric. Food Chem. 2016, 64, 7882−7889