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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX-XXX

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Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco Zhen Cao,†,‡,⊥ Wei Zhang,†,⊥ Xiangxue Ning,† Baomin Wang,*,† Yunjun Liu,*,§ and Qing X. Li*,‡ †

College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States § Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China ‡

ABSTRACT: Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize. KEYWORDS: Cry1Ac, Cry1Ia1, Cry1Ie, GMO, Western blot, linear epitope, antibody

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INTRODUCTION Bacillus thuringiensis (Bt) produces insecticidal crystal proteins named Cry or Cyt during sporulation.1 The genes encoding Bt insecticidal proteins were the most popularly implemented in genetically modified (GM) crops like cotton, maize, potato, and rice.2,3 There are different Cry protein classes. For example, Bt Cry1Ac protein is one of the δ-endotoxins being toxic to lepidopteran and dipteran larvae.4 Bt Cry1Ia1 (CryV) is another δ-endotoxin entomocidal to coleopteran and lepidopteran larvae.5 The GMO potato containing a modified Cry1Ia1 gene showed resistant to potato tuber moth (Phthorimaea operculella).6 The E. coli expressed Cry1Ie protein is also toxic to some lepidopteran like diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia f urnacalis), and soybean pod borer (Leguminivora glycinivorella).7 Cry1Ac gene has been widely employed in commercial GMO cotton and maize. To minimize the development of Bt resistance, Cry1Ie gene was modified and has been transferred in tobacco and maize, because less cross-resistance exists between Cry1Ie and Cry1Ac.8,9 Introducing different genes is a strategy for sustainable usage of Bt crops, as stacked traits GMO increased in recent years and accounted for 41% of the global acreage in 2016.10 Commercial GMO crops have been planted since 1996. The acreage of GMO crops increased steadily and reached 185.1 million hectares globally in 2016.10 Bt GMO crops have economically reduced the use of chemical pesticides and yielded quality produce. However, insect resistance to insecticidal toxins, toxicity to nontarget organisms, and threats to ecological balance are controversial concerns of GMO crops.11−14 Clarification of the misinformation and implementation of safety regulation will help GMO crop adoption globally.15 Enzyme-linked immunosorbent assays (ELISAs) and © XXXX American Chemical Society

its derived assays are sensitive, high-throughput, economic, and fast tools for GMO Bt protein detection, expression level measurement, and safety assessment, yet sometimes provide false-positive results.16−18 Western blot is widely used across a broad range of biological science disciplines19−22 as well as the detection of GMO proteins expression level, environmental fate or biosafety investigation.2,3,23 Western blot provides useful information, like protein degradation, truncation, and modification after translation, which is not readily gathered from other immunoassays. After gel electrophoretic separation and protein transfer, a target protein is detected by a specific antibody, which confirms the identity of the target protein. The specific binding of an antibody makes Western blot well-suited for detecting a target GMO protein and evaluating levels of intact protein expression. Both monoclonal antibody (mAb) and polyclonal antibody (pAb) have been used in Western blot. Antibody specificity and affinity with a target protein are essential characteristics. Antibody specificity allows minimizing background noises, while antibody affinity allows detecting the target protein at a very low concentration. Antibody specificity is often expressed with the cross-reactivity of the assay, while antibody affinity is often expressed with the assay sensitivity such as limit of detection and half-maximum inhibition concentration (IC50).24 An antiserum (containing pAbs) recognizing multiple epitopes is often more sensitive, less costly than a monoclonal Received: Revised: Accepted: Published: A

July 24, 2017 October 25, 2017 October 25, 2017 October 25, 2017 DOI: 10.1021/acs.jafc.7b03426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Apparatus. Cell culture plates and 96-well polystyrene microtiter plates were purchased from Costar (Corning, NY). A direct heat CO2 incubator and a microplate reader were purchased from Thermo (Franklin, MA). An electric heating constant-temperature incubator was purchased from Tianjin Zhonghuan Experiment Electric Stove Co. Ltd. An ultrasonic cleaner (KH-500E, Jiangsu, China) was purchased from Kunshan Hechuang Ultrasonic Apparatus Co. Ltd. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyvinylidene difluoride (PVDF) membrane and Western Blotting apparatus were purchased from Bio-Rad (Hercules, CA). Media. DMEM containing 10−20% (v/v) FBS was supplemented with 0.2 M glutamine, 50000 units/L penicillin, and 50 mg/L streptomycin. The standard medium was used for growing of myeloma and hybridoma cells. HAT selection medium was standard medium with 1% HAT medium supplements. Buffers and Solutions. Buffers and solutions included: (1) coating buffer (0.05 M Na2CO3−NaHCO3 buffer, pH 9.6); (2) phosphate-buffered saline (PBS) (0.01 M phosphate buffer containing 0.9% NaCl, pH 7.5); (3) block solution (10% skimmed milk powder in PBS); (4) PBS with 0.1% (v/v) Tween-20 (PBST); (5) PBST containing 0.5% (w/v) gelatin (PBSTG); (6) 3,3′,5,5′-tetramethylbenzidine (TMB) substrate buffer (Sigma-Aldrich); (7) stop solution (1 M HCl); (8) 2X protein loading buffer (20% glycerol (v/v), 10% beta-mercaptoethanol (v/v), 8% SDS (w/v), 0.1 M Tris-HCl, 0.025% bromophenol blue (w/v), pH 6.8); (9) electrophoretic transfer buffer (6.05 g of Tris base and 28.83 g of glycine dissolved in 1600 mL ddH2O and then add 400 mL methanol); (10) Tris-HCl buffered saline with 0.1% (v/v) Tween-20 (TBST) (0.1 M Tris−HCl buffer containing 0.1 M NaCl, pH 7.5).34 Myeloma Cell Line and Experimental Animals. The HATsensitive BALB/c mouse myeloma cell line named SP2/0-Ag14 obtained from the Chinese Institute of Veterinary Drug Control (Beijing, China) was applied to cell fusion experiments. The white New Zealand rabbit and female BALB/c mice were purchased from the Laboratory Animal Center of the Institute of Genetics (Beijing, China). All animal experiments were approved by Beijing Experimental Animal Management Office and performed in compliance with the regulation of Animals Welfare Act of the U.S. Department of Agriculture. All animals were raised under controlled temperature (22 ± 2 °C) and light (12 h light/12 h darkness) with air circulation, fresh water, and animal feed. Immunogen Preparation. A natural linear epitope commonly exists and may be not affected by protein denaturation.22 In order to expose more linear epitopes, Cry1Ac protein was denatured in 100 °C water bath. The optimal time duration of heat treatment was assessed to avoid the protein degradation. Bt Cry1Ac protein, dissolved in 10 mM PBS (pH 7.5) buffer at a concentration of 1.0 μg/mL, was incubated in boiling water for 0, 5, 10, and 20 min and then analyzed with ELISA kit and SDS-PAGE, respectively. For SDS-PAGE analysis, after heat treatment, the Bt Cry1Ac protein solution was mixed with 2× loading buffer (1:1, v/v) and directly loaded to the gel. Bt Cry1Ia1 (GenBank accession number Q45752) and Cry1Ie (GenBank accession number AAG43526) proteins were expressed in E. coli strain Transetta (DE3) and purified according to the described method.35 Cry1Ia1 and Cry1Ie were not treated at 100 °C because they were purified after cell lysis. The cell lysis reagents and conditions can promote random folding or partial denaturation of the proteins. An aliquot of 1 mL 1.0 mg/mL of Cry1Ia1 or Cry1Ie dissolved in PBS was mixed with 1 mL Freund’s adjuvant as immunogen, respectively. With the results of denaturation study, in practice, 1.0 mg/mL of Bt Cry1Ac protein solution after 5 min incubation in boiling water was cooled down at room temperature, then 1:1 mixed with Freund’s adjuvant as immunogen. Animal Immunization and Establishment of Mouse Hybridoma. Two New Zealand rabbits (3 kg) and five female BALB/c mice, 8 weeks old, were immunized with the immunogen. The protocols of immunization, antisera collection, cell fusion, antibody production, purification, mAb epitope determination were the same as described previously.34,36 The coating antigen was immobilized on the solid phase in direct ELISA similarly as antigen-PVDF membrane

antibody. However, a large diversity among animal individuals governs the antibody polymorphism. In antisera, only 0.5−5% antibodies specifically bind to the target protein.25 Animal individual diversity includes genetic variations, developmental differences, ages, and body sizes.26 pAbs are, therefore, heterologous and may have high background noise and high cross-reactivity, which is a main disadvantage when they are used to detect specific proteins in stacked traits (e.g., Bt Cry1Ac and Bt Cry1Ab) GMO crops. The introduction of mAb much overcomes these problems. Hybridoma technology allows to produce a specific antibody against a single antigenic determinant and to reproduce it indefinitely. mAbs are, therefore, homologous, have low cross reactivity and background interferences, and make reproducible Western blot assays. Procedures for evoking mAb suitable for Western blot are not commonly available in the literature. It is reported that mAb recognizing natural proteins used for ELISAs would not be necessarily applicable to Western blot.27,28 Solid surface adsorption of protein molecules in Western blot may induce conformation change or denaturation, especially significant changes in structure.29,30 Liu et al.21 obtained four mAbs that recognized West Nile virus, of which one mAb strongly recognized the native antigen in direct ELISA, but did not recognize it in Western blot. The conformational or discontinuous epitopes of natural target proteins are usually interrupted by denaturation.30 Modifications like deamidation during food processing may also change protein epitopes, which affects antibody recongnition.31 Partial renaturation happens during the electrophoretic transfer step of Western blot. Luo et al.33 found that mAb15-13 recognized a linear epitope of G protein, if tryptophan was replaced with arginine at position 251 on G protein, the mAb had no activity in Western blot.32 According to the 2011 statistics in the United States alone, an average annual amount of $350 million was wasted on ineffective antibodies, while as much as $800 million per year was wasted in the world, accounting for 50% of the total cost of global research antibody.25 The present study was designed to find a simple, economical, and reproducible method to produce mAb recognizing Cry1Ac, Cry1Ia1, or Cry1Ie for Western blot analysis.

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MATERIALS AND METHODS

Reagents. Bt Cry1Ac protein and ELISA Kit (Evirologix) bought from YouLong Biotech (Shanghai, China). E. coli expressed Bt Cry1Ia1 and Cry1Ie proteins were prepared in the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. ImmunoPure Monoclonal Antibody Isotyping Kit was purchased from Pierce (HRP/ABTS, Pierce, Rockford, IL, USA). Reagents purchased from Sigma-Aldrich (St. Louis, MO) included complete and incomplete Freund’s adjuvant, cell freezing medium dimethyl sulfoxide (DMSO, serum-free), polyethylene glycol (PEG) 2000, HAT (hypoxanthine, aminopterin, and thymidine)/HT (hypoxanthine and thymidine) medium supplements, L-glutamine, penicillin, streptomycin, mouse monoclonal antibody isotyping reagents, and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS). 3,3′-Diaminobenzidine (DAB) HRP color development kit was purchased from Tiangen Biotech Co. Ltd. (Beijing, China). Cell culture medium (Dulbecco’s modified Eagle’s medium, DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (PaisLey, Scotland). Goat antimouse IgG−horseradish peroxidase (IgG−HRP) was purchased from Jackson Immunoresearch Laboratories (West Grove, PA). All other reagents were purchased from Beijing Chemical Reagents Co. (Beijing, China). B

DOI: 10.1021/acs.jafc.7b03426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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those in the direct ELISA. For positive hybridoma screening, 100 μL of Bt Cry1Ac (100 ng/mL) were added to each well. For standard curve, 100 μL of 1000, 500, 250, 125, 62.5, 31.25, 15.6, and 0 ng/mL Bt Cry1Ac were added to each well, respectively. After incubation at 37 °C for 30 min and then washing, 100 μL of hybridoma supernatants or mAb were added and incubated at 37 °C for 30 min. For hybridoma screening, 100 μL/well of a diluted positive control (immunized mouse serum), a negative control (unimmunized mouse serum), or a blank control were also added. The next steps of IgG-HRP application, color development and so on were the same as those in the direct ELISA. Absorbance values at least 5 times higher than the negative control were considered positive. Sandwich ELISA for Cry1Ia1 and Cry1Ie was the same as above.35 GMO Bt Protein Extraction. GMO Bt Cry1Ac was extracted from transgenic cotton seeds (noncommercialized breeding material). Cotton seed shell was removed and the kernels were ground into powder. Ten grams of powder were suspended in 100 mL of 10 mM CAPS buffer, pH 10.5. Dissolved proteins were extracted at 4 °C for 4 h under magnetic bar stirring and followed by centrifugation at 11000g for 20 min. After lipid removal, the supernatant was collected. Solid ammonium sulfate was used to precipitate target proteins. Ammonium sulfate powder was slowly added into the supernatant with magnetic bar stirring in an ice bath until (NH4)2SO4 reached 60% (W/V) of the total volume. After another 30 min standing at 4 °C, the mixture was centrifuged at 4000g at 4 °C for 20 min. The precipitation was dissolved with 2 mL ddH2O and its pH was adjusted to 10.5 with 1 M of Tris base, followed by dialysis (molecular weight cutoff, 12 KD) with 10 mM PBS buffer, pH 7.5 at 4 °C. The dialysis buffer was changed every 6 h for a total of 6 changes. Protein from non-GMO cotton seed was extracted as described above for the negative control. GMO Bt Cry1Ie was extracted from GMO maize leaves.35 Maize (0.2 g) seedlings was ground on ice bath and then extracted with 5.0 mL PBST at 4 °C for 3 h. The mixture was centrifuged at 8000g for 10 min at 4 °C. The supernatant was diluted 10-fold in PBST when used for sandwich ELISA. Transgenic tobacco plants overexpressing Cry1Ia1 were generated (unpublished data) and Bt Cry1Ia1 protein was extracted from transgenic tobacco leaves. Tobacco leaves (0.3 g) was ground with 3 mL PBS and extracted with the same protocol as maize seedlings. Proteins used for false positive hybridoma counter screening were extracted from non-GMO tobacco leaves or maize seedlings with the same protocol, respectively. The protein concentration of the immunogen was determined with the Bio-Rad Protein Assay Kit (Hercules, CA, USA), with BSA as the standard. Prior to SDS-PAGE analysis, the protein concentration was diluted to 1.0 mg/mL with PBS buffer. SDS-PAGE and Western Blot. Both SDS-PAGE and native polyacrylamide gel electrophoresis were used to separate the protein prior to protein transfer. However, SDS-PAGE has high resolution and is wildly used.38 The Bt proteins were analyzed by SDS-PAGE. An aliquot of 15 μL of Bt protein was mixed with 2× loading buffer (1:1, v/v), after incubation for 5 min in boiling water and brief centrifugation, 13 μL of sample were loaded to each well. The SDSPAGE was performed with 5% stacking gel and 12% running gel.39 The gel was run at 80 V for 20 min and then 100 V for 90 min. The protein gel was stained with Coomassie Brilliant Blue R-250 or was transferred electrophoretically (1 h at 100 V) from the gel to PVDF membranes for Western blot analysis. The molecular weights of Bt proteins were estimated with prestained 10−170 KD protein ladders (Tayingfang Tech. Co., Ltd., Beijing, China). Western blot assays were performed as previously described.39 All incubation and washing steps were performed on a shaker at room temperature unless otherwise stated. After electrophoretic transfer, the PVDF membrane was washed with ddH2O three times and then blocked with blocking solution for 1 h. The membrane was then incubated with the mAb in blocking solution (1 μg/mL) overnight at 4 °C. The membrane was washed with TBST twice, 5 min per time. Goat antimouse IgG-HRP (1 μg/mL) in TBST was incubated with the membrane for 1 h. After three washes with TBST, the immunoreactive sites of the membrane were detected with DAB reagent (1 mL buffer contains 50 μL A and 50 μL B) in a clean glass dish.

adsorption.37 Direct ELISA was commonly applied to mAb screening. However, sandwich ELISA can form monolayer of antigen and better displays most epitopes to the primary antibody than direct ELISA. Therefore, direct ELISA (Figure 1a) and sandwich ELISA (Figure 1b)

Figure 1. ELISAs used for antisera titer testing or monoclonal antibody screening. (a) Direct ELISA; (b) Sandwich ELISA. were both used to screen positive hybridomas after cell fusion. To obtain an antibody recognizing a linear epitope as well as a conformational epitope, both semidenatured and natural GMO Bt proteins were used as the target proteins for mAb selection. The positive hybridomas that did not cross-react with nontarget proteins were selected, cloned by limiting dilution, and then expanded. The nontarget proteins were the soluble proteins extracted from non-GMO cotton seeds, tobacco leaves, and maize leaves. Rabbit polyclonal antisera were used as a capture antibody for sandwich ELISA. A New Zealand rabbit was immunized via subcutaneous injection on the back and inner thigh muscles of legs with 1 mg of immunogen in 1 mL of PBS emulsified with an equal volume (1 mL) of complete Freund’s adjuvant. Booster injections were carried out for three times at 3 weeks interval using incomplete Freund’s adjuvant. One week after the third injection, the rabbit antisera were collected from ear marginal vein for the anti-Bt protein titer testing with direct ELISA. Five female BALB/c mice were immunized subcutaneously with 100 μg/mouse of the immunogen emulsified with an equal volume of Freund’s complete adjuvant. Three booster injections prepared in the same manner using Freund’s incomplete adjuvant were carried out to each mouse at 2 weeks intervals. Test sera from the mice were collected 3 days after each boosting from the retrobulbar plexus. The titers of the sera were determined by direct ELISA and sandwich ELISA. The mouse showing the highest titer was injected intraperitoneally with 100 μg of the immunogen in 200 μL of PBS 3 days prior to fusion. Direct ELISA. The direct ELISA used for ascites and hybridoma supernatants titer tests was tests was performed as follows. An aliquot of 100 μL of 100 ng/mL Cry1Ac (or 200 ng/mL Cry1Ia1, Cry1Ie) protein diluted in coating buffer was pipetted into each well of microtiter plate and incubated at 37 °C for 3 h. Plates were washed three times with PBST buffer and blocked for 2 h at 37 °C with blocking solution. After 100 μL of diluted antisera or hybridoma supernatants were added to each well and incubated for 30 min at 37 °C, unbound compounds were removed by washing with PBSTG three times. A total of 100 μL goat antimouse IgG-HRP (1 mg/mL) conjugate diluted with PBST [1:1000 (V/V)] was added to each well for 30 min at 37 °C, followed by three washes with PBSTG. An aliquot of 100 μL of TMB substrate solution was then added to each well for enzymatic reaction, which was stopped after 15 min of incubation at room temperature by adding 100 μL of stop solution. Absorbance values were read at 450 nm with microplate reader. Sandwich ELISA. The sandwich ELISA was performed as follows. An aliquot of 100 μL of rabbit antisera (1:500 dilution with coating buffer) was added to each well of microtiter plate and incubated at 37 °C for 3 h. The washing and blocking steps were as the same with C

DOI: 10.1021/acs.jafc.7b03426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION

treatment lasted, the lesser natural epitopes remained. In order to further study the loss of recognition, SDS-PAGE was applied to analyze the heat semidenatured protein. Figure 2c shows that Cry1Ac (around 67 KD) did not totally disappear after 20 min of 100 °C water bath treatment. However, the band density decreased to 56% in 10 min and 46% in 20 min (Figure 2d). These may be due to protein degradation or precipitation during heat treatment. It was observed that treatment of 1 mg/ mL of Bt Cry1Ac protein in boiling water for 10 min caused notable precipitation of the protein. In order to expose more continuous epitope and yet maintain solubility and integrity of Cry1Ac as high as possible, treatment in a 100 °C water bath for 5 min was used to prepare the immunogen for animal immunization. Negligible precipitation of Cry1Ac was observed after the treatment at 100 °C for 5 min. It is noteworthy that completely denatured proteins are not soluble in water and organic solvent.40 Such difficulty is often not recognized during antibody production. When Cry1Ac was treated in a 100 °C water bath for a long period of time, the heat denatured Cry1Ac was not dissolved or mixed well in the complete and incomplete Freund’s adjuvant. The best treatment time or temperature depends on the protein concentrations and properties. Antibody Generation and Characterization. After 3 boosts, the rabbit antisera were tested with direct ELISA. The titer of the positive rabbit antiserum was above 1 × 103. The mouse of which the titer was greater than 6.4 × 104 was used for cell fusion (data not shown). Both direct ELISA and sandwich ELISA were used for Cry1Ac positive hybridoma screening after cell fusion. Direct ELISA was used for Cry1Ia1 positive hybridoma screening, sandwich ELISA was used to validate those mAb. For Cry1Ie positive hybridoma selection, only sandwich ELISA was used. Supernatants of hybridoma 9 days after cell fusion were screened to identify positive clones. Twenty five positive monoclonal hybridomas for Cry1Ac were identified with direct ELISA in the first screening. Finally, after counter screening with proteins extracted from non-GMO cotton seeds, two monoclonal hybridomas that could continuously secret antibody were obtained, named 1F2 and 3C9. With sandwich ELISA screening, 11 positive hybridomas against Cry1Ac were obtained at the beginning. Counter screening was also implemented to identify false positive antibodies that crossreacted with nontarget proteins. At the end of the screening, hybridomas 5E9C9 and 3E6E2 were chosen for ascites production. The mAb for Cry1Ac was purified from mouse ascites and then dialyzed against PBS.34 The concentration of mAb 1F2, 3C9, 3E6E2, and 5E9C6 were 0.54, 1.32, 0.46, and 1.74 mg/mL, respectively. The mAb 1F2 and 3C9, which were screened with direct ELISA, could not be implemented to sandwich ELISA as recognizing primary antibody (data not shown). Such differential performance of the same mAb in two assay formats may be caused by the epitope configuration changes after antigen-solid phase immobilization in the direct ELISA. Both 3E6E2 and 5E9C6 showed dose response to the concentration of heat semidenatured Cry1Ac. With mAb 3E6E2 as the primary antibody at 1 μg/mL, the standard curve fit well in an equation of absorbance = 1.721/ (1+exp (−0.0211*(C-96.188))), R2 = 0.9995, where C is concentrations of Cry1Ac in ng/mL (Figure 3). The working range of the standard curve was 15.6−1000 ng/mL. 5E9C6 and 3E6E2 had no reaction with natural or heat semidenatured Cry1Ac in direct ELISA. The specificity of the obtained mAb varied each

Immunogen Preparation of Cry1Ac. After 10 min of treatment in a 100 °C water bath, the protein solution was diluted 10 times with PBS for ELISA tests. The heat semidenatured Bt Cry1Ac protein (100 ng/mL) lost recognition by the commercial ELISA assay that detected the natural Cry1Ac (Figure 2a). After 5 min of heat treatment, the optical density at 450 nm (OD450) of 100 ng/mL Bt Cry1Ac detected by the ELISA kit decreased to approximately one tenth (from 3.759 to 0.387) (Figure 1b). The results indicate that the epitope of Cry1Ac, which could be recognized by the antibody, was interrupted by heat treatment because of denaturation, degradation, or precipitation of Cry1Ac. As the longer the heat

Figure 2. ELISA and SDS-PAGE results of Bt Cry1Ac protein treated under varying time periods in a boiling water bath. (a) After 10 min treatment with a 100 °C water bath, Bt Cry1Ac protein could not be detected by the ELISA kit. Each sample was measured in triplicate. (b) After 5 min treatment, the OD450 of 100 ng/mL Bt Cry1Ac detected by the ELISA kit decreased from 3.759 to 0.387. (c) SDS-PAGE results of Bt Cry1Ac standard protein with 0, 10, and 20 min boiling water bath treatment. (d) Densitometric analysis of Bt Cry1Ac band of SDS-PAGE. Optical density was normalized to 0 min treatment. D

DOI: 10.1021/acs.jafc.7b03426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 2. All Six Hybridoma Cell Lines (i.e., mAbs) Selected by dELISA Well Recognized Both Natural Cry1Ia1 and Semidenatured Cry1Ia1, but Could Not Be Used in Western blot recognition for the target protein mAb (subclass) 3B21F7 (IgG2b) 2B62C4 (IgG1) 4F81F5 (IgG2b) 1G101H7 (IgG1) 2A71E3 (IgG1) 2E51F7 (IgG1)

Figure 3. A standard curve of heat semidenatured Bt Cry1Ac protein detected with sandwich ELISA. Each value represents the mean of three replicates.

dELISA dELISA sELISA sELISA

semidenatured Cry1Aca

usability of the mAbs in Western blot

1F2 (IgG1) 3C9 (IgG2b) 5E9C6 (IgG2a) 3E6E2 (IgG2a)

++++b

++

not applicable

++++

-

not applicable

+

+++

not applicable

-

+++

applicable

++b

+++

not applicable

+++

++++

not applicable

++++

++++

not applicable

+++

+++

not applicable

+++

++++

not applicable

++++

++++

not applicable

Fifteen hybridomas showed positive reaction with Cry1Ie at the beginning. Sandwich ELISA selected four hybridomas, of which the mAb had moderate or strong reaction with Cry1Ie, but only one could continuously secret antibody (1G42D6, subclass IgG1). mAb 1G42D6 had strong reaction with the natural Cry1Ie and had very strong reaction with semidenatured Cry1Ie in sandwich ELISA. SDS-PAGE and Western Blot. The concentration of Cry1Ac in the protein extracts from GMO cotton seed was quantitatively determined with the ELISA kit. The concentration of Cry1Ac in the seed was estimated to be approximately 10 μg/g wet weight seeds (moisture content 8.6%). The concentration of total protein in the protein extracts from GMO seed was approximately 25 mg/g, and after ammonium sulfate precipitation, the concentration of total protein was adjusted to 10 mg/mL with PBS. SDS-PAGE (Figure 4a) shows a single band of Cry1Ac around 67 KD on lane 1. No band could be observed on lane 2 because the

recognition for the target protein

natural Cry1Aca

usability of the mAbs in Western blot

Natural Cry1Ia1 was a protein solution extract from GMO tobacco leaves. Semidenatured Cry1Ia1 was purified from E. coli cell lysis. b+, weak recognition (0.2 ≤ OD < 0.5); ++, moderate recognition (0.5 ≤ OD < 1.0); +++, strong recognition (1 ≤ OD < 2.0); ++++, very strong recognition (OD ≥ 2.0); -, no recognition (OD < 0.2).

Table 1. Comparison of Using the Direct ELISA (dELISA) and Sandwich ELISA (sELISA) To Screen Hybridoma Cell Lines (i.e., mAbs) for Their Recognition of Natural Cry1Ac and Semidenatured Cry1Ac and Their Usability in Western Blot

mAb (subclass)

semidenatured Cry1Ia1a

a

other (Table 1). Both 1F2 and 3C9 very strongly recognized natural Cry1Ac in the direct ELISA, whereas 1F2 had only

assay used to screen hybridoma cell lines

natural Cry1Ia1a

a

Natural Cry1Ac was not heat treated protein. Semidenatured Cry1Ac was Cry1Ac treated in a water bath at 100 °C for 5 min. b+, weak recognition (0.2 ≤ OD < 0.5); + +, moderate recognition (0.5 ≤ OD < 1.0); +++, strong recognition (1 ≤ OD < 2.0); ++++, very strong recognition (OD ≥ 2.0); -, no recognition (OD < 0.2).

moderate reaction with heat semidenatured Cry1Ac, and 3C9 did not react with the immobilized heat semidenatured Cry1Ac in the direct ELISA at all. The results indicated that heatdenaturation may cause different or more conformation change than denaturation caused by solid-phase absorption. Both 5E9C6 and 3E6E2 had strong reaction with heat semidenatured Cry1Ac in the sandwich ELISA, but only 5E9C6 had weak reaction with the natural Cry1Ac (Table 1). The antisera titers of mice were higher than 6.4 × 104 after three booster immunization with Cry1Ia1 immunogen. Six positive hybridomas were obtained via direct ELISA screening (Table 2). mAb 3B21F7 moderately recognized the natural Cry1Ia1 from tobacco, while the other 5 mAbs strongly recognized the natural Cry1Ia1. All six mAb had strong or very strong recognition with semidenatured Cry1Ia1 in direct ELISA.

Figure 4. Comparison of SDS-PAGE and Western blot results of Bt Cry1Ac protein. Lane 1, Marker; lane 2, 1 mg/mL Bt Cry1Ac standard protein; lane 3, 10 μg/mL Bt Cry1Ac standard protein; lane 4, blank control; lane 5, Bt Cry1Ac purified from GMO cotton seeds. SDSPAGE was run in one piece of gel and cut into two parts; one was stained with Coomassie brilliant blue (a), the other one was used for Western blot with mAb 3E6E2 (b). E

DOI: 10.1021/acs.jafc.7b03426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

selected mAbs recognizing Cry1Ac could be used for Western blot and the only one mAb for Cry1Ie selected with the sandwich ELISA was also successfully applied to Western blot.35 mAbs have advantages to exclude false positives during hybridoma counter screening. mAbs reduce cross reactivity because of the specificity. mAb 3E6E2 could recognize heat semidenatured Cry1Ac, but not the natural Cry1Ac in sandwich ELISA. This suggested that the mAb 3E6E2 may recognize linear epitope after Cry1Ac protein denaturation. Such denaturation could be caused by heat treatment or chemical reagent like urea. Because protein-solid phase adsorption can cause denaturation or conformation changes of the protein, the two mAbs for Cry1Ac and the six mAbs for Cry1Ia1, which were screened with direct ELISA, could not be applied to Western blot. The coated capture antibody in sandwich ELISA displayed the target protein by forming a mono layer, which is probably easier for antibody binding to the specific epitope. The results of the present study suggest that semidenatured protein shall be a good immunogen for developing mAb or antisera for Western blot utility. Sandwich ELISA is more efficient than direct ELISA to screen positive monoclonal hybridoma for applications in Western blot.

amount of protein was under the limit of detection of Coomassie brilliant blue staining. The four mAb were studied for Western blot applications at different concentrations. 3E6E2 (1 μg/mL) successfully detected both the prokaryotic expression Cry1Ac and cotton seed expressed Cry1Ac (Figure 4b). One single band could be observed on lane 2, which means that Western blot is more sensitive than SDS-PAGE for protein analysis. For lane 5, band around 67 KD was clearly observed; however, there were one larger band around 70 KD and other two smaller bands near 40 KD (Figure 4b). Western blot is a powerful tool for transgenic protein safety evaluations as molecular weight difference could be revealed after protein modification. Equivalency tests of Cry1Ia1 from GMO potato and E. coli produced Cry1Ia1 showed no difference in size in Western blot.23 The Western blot results also showed that Cry2Aa2 expressed in tobacco leaves was larger than the E. coli expressed Cry2Aa2.12 The larger molecular protein on lane 5 could be the cotton Cry1Ac protein precursor. The small molecular protein could be either the degraded peptides during extraction or another small protein bearing the epitope that could be recognized by the antibody.41 Western blot assay could be used to study tissuespecific protein expression due to different post-translation modification.42 The result of the present study indicated that Western blot could give more information on protein heterogeneity than ELISAs because Western blot employs the electrophoresis separation power and antibody’s high specificity and affinity. However, monoclonal antibody recognizing one epitope may not recognize some truncated or degraded proteins of which the epitope is lost, which cause false negative results. The proteins of those bands require further characterization. The SDS-PAGE and Western blot of Cry1Ia1 and Cry1Ie were conducted as described previously.35 None of the six mAb of Cry1Ia1 was successfully implemented to Western blot as there was no signal (data not shown). There was only one band when 1G42D6 was used as the antibody for the Western blot analysis of Cry1Ie from GMO maize leaf extraction.35 These results indicate that mAb 1G42D6 may bind to the natural linear epitope of Cry1Ie and have high specificity. The natural linear epitope was normally formed by hydrophilic amino acid group on the surface of natural protein and keep stable even protein denaturation.22 This would be a plausible explanation for which why mAb 1G42D6 could be applicable to sandwich ELISA as well as Western blot. In summary, all mAbs selected with the direct ELISA could not be implemented to Western blot (Table 3). The number of positive hybridomas selected with the sandwich ELISA was less than that with the direct ELISA. However, one of the two

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*Tel.: +8601062731305; Fax: +8601062732567; E-mail: [email protected]. *Tel.: +86010-82105863; Fax: +86010-82105863; E-mail: [email protected]. *Tel.:+1 808 956 2011; Fax: +1 808 956 2011; E-mail: qingl@ hawaii.edu. ORCID

Qing X. Li: 0000-0003-4589-2869 Author Contributions ⊥

This work was funded by the National Natural Science Foundation of China (31271649) and the US National Institutes of Health Research Centers in Minority Institutions Program (G12 MD007601). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Hejian Guoxin Cotton Profession Cooperative, Hebei, China for providing the GMO cotton seeds. ABBREVIATIONS USED Bt, Bacillus thuringiensis; GMO, genetically modified organisms; mAb, monoclonal antibody; pAb, polyclonal antibody; ELISA, enzyme-linked immunosorbent assay; dELISA, direct ELISA; sELISA, sandwich ELISA; DMSO, dimethyl sulfoxide; PEG, polyethylene glycol; HAT, hypoxanthine, aminopterin, and thymidine; HT, hypoxanthine and thymidine; CAPS, Ncyclohexyl-3-aminopropanesulfonic acid; DAB, 3,3′-diaminobenzidine; IgG-HRP, IgG-horseradish peroxidase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PBST, PBS with 0.1% (v/v) Tween-20; PBSTG, PBST containing 0.5% (w/v) gelatin; TBST, Tris-HCl buffered saline with 0.1% (v/v) Tween-20; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electro-

target proteins

direct ELISA sandwich ELISA

usability of mAb

Cry1Ac

Cry1Ia1

no. of positive mAb screened no. of mAb that were applicable in Western blot no. of positive mAb screened no. of mAb that were applicable in Western blot

2 0

6 0

2 1

Z.C. and W.Z. contributed equally to this work.

Funding

Table 3. mAb Applicable in Western Blot Out of the Positive mAb (i.e., Hybridoma Cell Lines) Screened by Direct ELISA and Sandwich ELISA Against Target Bt Proteins

screening assay

AUTHOR INFORMATION

Corresponding Authors

Cry1Ie

1 1

F

DOI: 10.1021/acs.jafc.7b03426 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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phoresis; PVDF, polyvinylidene difluoride; TMB, 3,3′,5,5′tetramethylbenzidine

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