Development of a Rapid Immunochromatographic Lateral Flow Device

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Development of a Rapid Immunochromatographic Lateral Flow Device Capable of Differentiating Phytase Expressed from Recombinant Aspergillus niger phyA2 and Genetically Modified Corn Xiaojin Zhou,† Elizabeth Hui,‡ Xiao-Lin Yu,∥ Zhen Lin,§ Ling-Kui Pu,† Zhiguan Tu,∥ Jun Zhang,‡,# Qi Liu,#,⊥ Jian Zheng,*,§ and Juan Zhang*,Δ

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Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Beijing 100081, People’s Republic of China ‡ Artron BioResearch Inc., 3938 North Fraser Way, Burnaby, British Columbia V5J 5H6, Canada ∥ Key Laboratory of Diagnostic Medicine and §Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 4000016, People’s Republic of China # Ji Nan Kangbo Biotechnology, 2711 Ying Xiu Road, Jinan, Shandong Province 250101, People’s Republic of China ⊥ Beijing Artron Jingbiao Biotech Inc., 19 Tianrong Street, Daxing Bio-medicine Industry Park, Daxing District, Beijing 102600, People’s Republic of China Δ Blood Transfusion Department, Second Affiliated Hospital of Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, People’s Republic of China ABSTRACT: Phytase is a phosphohydrolase considered highly specific for the degradation of phytate to release bound phosphorus for animal consumption and aid in the reduction of environmental nutrient loading. New sources of phytase have been sought that are economically and efficiently productive including the construction of genetically modified (GM) phytase products designed to bypass the costs associated with feed processing. Four monoclonal antibodies (EH10a, FA7, AF9a, and CC1) raised against recombinant Aspergillus niger phyA2 were used to develop a highly specific and sensitive immunochromatographic lateral flow device for rapid detection of transgenic phytase, such as in GM corn. Antibodies sequentially paired and tested along lateral flow strips showed that the EH10a−FA7 antibody pair was able to detect the recombinant yeast-phytase at 5 ng/mL, whereas the AF9a−CC1 antibody pair to GM phytase corn was able to detect at 2 ng/mL. Concurrent to this development, evidence was revealed which suggests that antibody binding sites may be glycosylated. KEYWORDS: phytase, glycosylation, monoclonal antibodies, lateral strips, GM corn

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INTRODUCTION Phytase (myo-inositol hexakisphosphate phosphohydrolase) EC 3.1.3.8 is part a class of phosphatases that can catalyze the sequential hydrolysis of phytate to lower phosphorylated inositol and inorganic phosphates.1,2 Phytate is the main storage form of phosphorus in livestock feed such as seeds and cereal grains, representing nearly 90% of their total phosphorus content.3,4 The digestive microbial faunas of monogastric animals lack the necessary phosphorus hydrolyzing enzymes, and as a result much undigested phytate-associated phosphorus is lost into the environment.5 This can lead to excessive phosphorus loading in soil and water, and the ensuing pollution can affect other ecosystems.1,6 Phytate is considered as an antinutrient due to its chelating ability, which results in the formation of complexes with nutritionally important multivalent metal ions, such as Fe3+, Zn2+, and Ca2+, making them unavailable for absorption.6,7 Animal feed is often supplemented with inorganic phosphorus or phytase additives to meet the nutritional requirement for growth by increasing phosphorus availability and aid in the reduction in phosphorus excretion.1,8 The subsequent development and application of transgenic phytase plants and animals have been shown as alternatives to exogenous additives, thereby reducing processing © 2015 American Chemical Society

and formulation costs and concerns of contaminates, such as fluorine and heavy metals, accrued during the manufacturing process of inorganic feed phosphates.6,8,9 Higher levels of phytate-degrading activity are achievable by optimizing expression of specific genetic events and through the use of economically competitive expression/secretion systems.6,11 Such phytase supplementation had greatly improved the phytate antinutrient factor, prompting the search for more temperature- and pH-tolerant phytases and propelled phytase optimization technology through genetic and protein engineering.8,10,11 The phytase commercial market volume has exceeded U.S. $250 million and is growing at around 10% per year.12 Phytases can be glycosylated, and the level of glycosylation is known to be highly variable.2,13,14 For example, Escherichia coli phytase is nonglycosylated,15 whereas the glycosylation pattern between different fungal phytases varies on the basis of the expression system16 and between individuals on a given expression system.2 Glycosylation can have many effects on Received: Revised: Accepted: Published: 4320

January 19, 2015 April 14, 2015 April 22, 2015 April 22, 2015 DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326

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Journal of Agricultural and Food Chemistry

Coast Seeds (WCS; two of F1, two of P, one of DF1, and one unknown), green bean, white bean, and seven generic varieties of corn (purchased from local markets)] were used in characterization studies. Phytase monoclonal antibodies (MAbs, EH10a, FA7, AF9a, and CC1) raised against a recombinant A. niger 963 (phyA2) phytase were donated by the Chinese Biotechnology Research Institute in Beijing and prepared in a manner similar to that described in Chen et al.24 Epitope Characterization. To characterize the epitopes recognized by the purified phytase MAbs, an ELISA and the additivity index (AI) described by Friguet et al.25 were used. Briefly, wells of a 96-well plate were coated with 100 μL of 2 μg/mL recombinant phytase and incubated overnight at 4 °C. The following day, the wells were incubated with 100 μL of antibodies, EH10a, FA7, AF9a, CC1, individually or in paired combinations (50 μL each) of equivalent concentrations at 1:1000 overnight at 4 °C. The following day, the wells were incubated with 100 μL of a goat anti-mouse IgG−HRP secondary antibody at 1:1000 for 30 min at 37 °C. The absorbance value for each treatment was recorded at 450 nm. The AI was calculated using the following equation: {[2A1+2/(A1 + A2)] − 1} × 100%, where A1, A2, and A1+2 are the absorbance values for the individual antibodies and the respective combined pairs. If the two antibodies are directed against different epitopes (no competition), A1+2 should be equal to the sum of A1 and A2 and the AI value should approach 100%. If the two antibodies are directed against the same epitope (competition), A1+2 should be equal to the mean value for A1 and A2 and AI should be close to 0%. The threshold was determined by AI ≥ 40%. Protein Analysis. Protein and Western blot analyses on the specificity of the MAbs were evaluated by 15% SDS-PAGE using 1 μg of recombinant phytase and 50 mg of ground seeds from GM phytase corn, generic corn, green bean, white bean, Pickseed2733 corn, WCS F1 corn, four varieties of GM corn, and one GM soybean variety. The phytase could be released from the GM phytase corn only by homogenizing the corn seeds in a modified Tris buffer [50 mM TrisHCl (pH 8.0), 10 mM KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM βmercaptoethanol, 0.1% BSA, 13% sucrose, and SigmaFAST Protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA)] at 400 μL per 100 mg tissue, after which the sample was centrifuged at 4500g for 10 min. Laemmli loading buffer26 was then added to the supernatant and incubated at 65 °C for 20 min. The remaining seeds were ground, homogenized in 500 μL of Laemmli buffer, boiled for 10 min, and centrifuged for 2 min at 12000g. To improve Western blot detection, 5 μg of recombinant phytase and 100 mg of ground seeds were used. Twenty microliters of each prepared seed supernatant was used for protein and Western blot analysis. The recombinant phytase, 20 μL of water, and 10 mM Tris (the latter two as negative controls) were also boiled in Laemmli buffer as described above. The membrane blot was incubated overnight at 4 °C with a combined mixture of EH10a, FA7, AF9a, and CC1 MAbs (each at 1:400), whereas the SDS-PAGE protein gel was stained with Coomassie Brilliant Blue. The following day the membrane was incubated with horseradish peroxidise conjugated goat anti-mouse immunoglobulin (IgG−HRP at 1:1000) for 90 min at room temperature. Proteins on the blot were developed using the 3,3′-diaminobenzidine tetrahydrochloride (DAB; SigmaAldrich) method with 0.1% hydrogen peroxide. To evaluate the nature of the MAbs directed to epitopes on phytase, an additional Western blot analysis was performed. Purified recombinant phytase protein isolated from yeast and ground seeds of GM phytase corn were both resuspended with PBS to a concentration of 200 μg/mL and stored at 4 °C until required. Every 2 weeks for a period of 10 weeks, 10 μL from each of the recombinant phytase and GM phytase corn was boiled with 10 μL of loading buffer for 5 min and loaded onto a 10% SDS-PAGE. Each transferred membrane blot was incubated overnight at 4 °C with a combined mixture of MAbs as described above. The subsequent steps are as described above, except the concentration of the IgG−HRP secondary antibody was 1:5000 with an incubation time of 30 min. Construction and Evaluation along Immunolateral Flow Test Strips. The detection phytase antibody (the test line antibody, either FA7 or CC1) and the goat anti-mouse IgG (the control line

the properties of a protein, such as on stability, solubility, and metabolic energy.2 Glycosylation of phytase has been shown to affect thermostability,13,17,18 which would be a concern regarding enzyme activity loss due to heat such as that from feed pelleting. Phytases for commercial use have been isolated mainly from fungi and bacteria,6,8,11 and selection for an efficacious product is greatly dependent on the source,1 tolerance to processing factors, digestive resistance, and production costs.6,8,19 Major feed crops have been genetically modified to increase phytase expression and are at various stages of product development.20 For example, two separate studies on transgenic phytase barley have both reached an advanced developmental stage complete with field trials; however, only transgenic phytase corn (Event B23-3-1) has received approval status for commercial sale. Integration of such phytases as transgenes in agricultural products in a growing industry would benefit from inclusion of a means to verify transgenic phytase presence in food nutritional and environmental management strategies wherein concerns of phytate mineral availability and environmental issues need to be assessed. Development of a rapid diagnostic test (RDT), which is a qualitative immunoassay (consisting of target specific antibodies) used in point-of-care testing, for transgenic phytase would offer an efficient, convenient, and affordable method of detection. Aspergillus niger phytase (phyA2), which has 10 potential Nglycosylation sites,2 was cloned and expressed in a methylotrophic yeast, Pichia pastoris.21 This same phytase was cloned into corn, yielding seeds with a maximal phytase activity of 125 FTU/g kernels, 1000-fold above that of the wild type, and with 1000 g of kernels containing up to 67 times the feed industry requirement.22 Corn is a globally important animal feed crop; in China, corn occupies 30 million hectares of land from which much is used to feed 500 million pigs (approximately 50% of the global herd) and 13 billion chickens, ducks, and poultry.23 As mentioned above, only transgenic phytase corn has been approved for commercialization; accessibility coupled with the ensuing demand for corn production, all make it an ideal product candidate for this project. Herein, two pairs of highly sensitive and specific monoclonal antibodies (MAbs) raised against A. niger recombinant phytase and generated through an extensive screening technique were described whereby each was found to detect a phytase protein of different sizes, possibly due to glycosylation. Using these MAbs, the transgenic corn was found to express lower levels of the larger-sized phytase than the recombinant phytase had expressed in yeast; however, the level of detection was predicated on the condition of the expressed phytase. Together, these findings provide important insight regarding the structural properties of phytase epitopes, suggesting a possible role of carbohydrates within antibody binding sites, and lead to the development of RDT strips coated with MAbs that can detect two differently sized proteins of transgenic phytase.

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MATERIALS AND METHODS

Recombinant Phytase, Seeds, and Antibodies. A. niger 963 phytase (phyA2) expressed in P. pastoris was isolated by and donated from the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences in Beijing, China. This recombinant protein was freeze-dried, reconstituted in PBS (at 200 μg/mL), and stored at 4 °C until required. This group also donated genetically modified (GM) phytase corn seeds, expressing this same A. niger phytase gene phyA2. Seeds from various plants [six varieties of GM corn, one variety of a GM soybean, corn (2733) from Pickseed, six corn varieties from West 4321

DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326

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Journal of Agricultural and Food Chemistry antibody) were diluted to a standard concentration of 1.5 mg/mL with 10 mM Tris-HCl (pH 8.0) and applied in a thin line onto a nitrocellulose membrane. The colloidal gold conjugated phytase capture antibody was prepared as described in Chen et al.24 Briefly, 1.4 mL of 1% Na3C6H5O7 was first added to 100 mL of boiled 0.01% HAuCl4. The colloidal gold solution was then allowed to cool gradually, after which the pH was adjusted to 8.4 with 1% K2CO3. The EH10a or AF9a antibodies were added dropwise into 10 mL of the colloidal gold solution and left to stabilize at 4 °C. After 30 min, BSA blocking agent was added, and the solution was placed at 4 °C for another 2 h. The mixture was centrifuged twice, the first time to recover the supernatant and the second to conjugate pellet, which was then suspended in 10 mM borax buffer (with 2% BSA and 0.05% NaN3, pH 8.0). The conjugated antibody (i.e., EH10a if the detection antibody was FA7 or AF9a if the detection antibody was CC1) was sprayed twice onto a sheet of fiberglass and dried at 37 °C. The phytase lateral flow test strips were assembled as described in Chen et al.24 Two grams of each seed variety was ground in 10 mM Tris-HCl. The absorption pad end of a prepared test strip was immersed in seed supernatant. Test strips were also immersed in 200 μg/mL of recombinant phytase, 200 μg/mL of transgenic corn, distilled water, and 10 mM Tris-HCl (the latter two as negative controls). A response was observed within 5 min. The appearance of two bands, one each at the test and control sites, represents a positive test result; a single band at the control site represents a negative test result. The absence of a line at the control site indicates the test is invalid. To determine the sensitivity of the antibodies to phytase along an immunolateral flow test strip, recombinant phytase was diluted in concentrations from 200 to 0.002 μg/mL by 10 mM Tris-HCl (pH 8.0) and tested using the EH10a−FA7 MAb match pair, whereas the transgenic phytase corn was tested in concentrations from 400 to 0.001 μg/mL using the AF9a−CC1 MAb match pair. The responses of the strips were evaluated as above. The nature of the MAbs directed to epitopes on phytase was evaluated along an immunolateral flow test strip. Individual test strips consisting of either the EH10a−FA7 or the AF9a−CC1 MAb match pairs were immersed in the reconstituted recombinant or GM corn phytase samples every 2 weeks for a period of 10 weeks. The samples were stored at 4 °C until required. With the “MAX” line on the test strip positioned above the liquid level, a sample was allowed to migrate halfway up the strip, after which the strip was removed. The results were obtained within 30 min, and the strips were evaluated as above. The stability of test strips was assessed when kept for 1 year at room temperature.

Evaluation of Monoclonal Antibody Pairs to Phytase Protein. P. pastoris is a methylotrophic yeast that, when under the control of a methanol-inducible alcohol oxidase (AOX1) promoter, has been successfully used to produce many proteins with varying degrees of success,27−29 and these proteins are often glycosylated.30 Protein analyses of various seed varieties (Figure 1A) were used to assess MAb specificity along with a

Figure 1. Monoclonal antibodies (MAbs) target phytase. (A) Fifty micrograms of plant seeds was ground in 500 μL of Laemmli buffer of which 20 μL was loaded into each lane. The lanes are as follows: (1) water; (2) 10 mM Tris-HCl, pH 8.0; (3) marker; (4) 1 μg of recombinant phytase; (5) GM phytase corn; (6) corn 1; (7) green bean; (8) white bean; (9) Pickseed2733 corn; (10) WCS F1 corn; (11) GM corn 1; (12) GM corn 2; (13) GM corn 3; (14) GM corn 4; (15) GM soybean. (B) Western blot analyses were conducted on the samples used in (A), except that 5 μg of recombinant phytase and 100 mg of ground plant seeds were used and probed with an equal mixture of MAbs EH10a, FA7, AF9a, and CC1. The protein marker sizes in kilodaltons (kD) are indicated on the left of the blot. GM, genetically modified; WCS, West Coast Seeds. Unless stipulated, GM corn samples are not GM phytase corn. Additional methods are described in the text.

recombinant A. niger phytase expressed from yeast and a genetically modified (GM) corn containing this same A. niger phytase. The samples were probed with a MAb mixture of EH10a, FA7, AF9a, and CC1 (in equal proportions). The recombinant A. niger phytase expressed from yeast shows a band of approximately 75 kDa (Figure 1B, lane 4), whereas the GM A. niger phytase corn reveals a band of about 60 kDa (Figure 1B, lane 5). Further, this MAb mixture did not crossreact with the other test seeds as shown from Western blot analysis (Figure 1B, lanes 6−15). The larger-sized protein from the yeast-expressed phytase may be due to the greater degree of glycosylation often observed in proteins expressed from yeast. Differences in glycosylation pattern expressed from different hosts may be attributed to the discrete processes found in their respective cellular environments. For example, A. fumigates was found to produce a phytase that can tolerate boiling at 100 °C for 20 min, retaining 90% of its initial activity.31 Lucca et al.32 found that when rice containing the A. f umigates phytase was boiled for 20 min, the enzyme had retained 50% of the enzyme activity. However, when rice seeds were cooked under the same conditions, only 8% of the phytase activity was retained. Similarly, these differences in thermotolerance may be attributed to the different glycosylation patterns in phytase expressed between diverse hosts. Phytases that have high

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RESULTS AND DISCUSSION Monoclonal Antibody Characterization. Phytase is considered the most prominent enzyme to hydrolyze phytate and release bound phosphorus in a form available to animals for growth and development. A recombinant A. niger phytase (phyA2) expressed in P. pastoris21,22 was used to produce MAbs employing a very extensive and novel screening technique.24 From a total of 976 hybridoma cell strains, 32 positive cell clones using repetitive ELISAs and cross-responsive screening of MAbs against A. niger (phyA2) phytase were generated (personal communication, Biotechnology Research Institute). Cell lines screened for the strongest positive signal in phytasechallenged cultures were selected, culminating in four MAbs candidates (EH10a, FA7, AF9a, and CC1). AI values were calculated to determine competition for the binding sites between the four MAbs. The following AI values were found for the following antibody pairs: FA7+AF9a (67.7%), FA7+CC1 (91.4%), AF9a+EH10a (57.7%), EH10a+CC1 (69.7%), FA7+EH10a (59.6%), and AF9a+CC1 (≥99%). These results indicate that any pair of these antibodies would result in the targeting of a different phytase epitope. 4322

DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326

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Journal of Agricultural and Food Chemistry

Figure 2. Both match pair EH10a−FA7 and AF9a−CC1antibodies show high specificity to phytase. (A) Phytase specificity analysis was conducted using lateral flow strips containing the EH10a conjugate antibody and the FA7 capture antibody. Strips 1−4 were immersed in a 10 mL solution consisting of (1) 200 μg/mL recombinant phytase, (2) 200 μg/mL transgenic phytase corn, (3) 10 mM Tris-HCl, and (4) distilled water. Strips 5− 28 were immersed in the supernatant of 2 g of seeds ground in 10 mL of 10 mM Tris-HCl for 1−5 min. The strips were tested using the following seeds: (5) corn 1; (6) corn 2; (7) corn 3; (8) corn 4; (9) GM corn 1; (10) GM soybean; (11) Pickseed2733 corn; (12) GM corn 2; (13) GM corn 3; (14) GM corn 4; (15) WCS F1 corn 1; (16) WCS F1 corn 2; (17) WCS P corn 1; (18) WCS P corn 2; (19) WCS DF1 corn; (20) green soybean; (21) WCS corn; (22) green bean; (23) white bean; (24) corn 5; (25) corn 6; (26) corn 7; (27) GM corn 5; (28) GM corn 6. For each strip the upper line is the control (C) line containing antibodies to goat anti-mouse IgG and the lower line (if present) is the test (T) line with the FA7 capture antibody. The corn samples used in strips 5−8 and 24−26 were generic (non-GM) varieties. (B) Phytase specificity analysis was conducted using lateral flow strips containing the AF9a conjugate antibody and the CC1 capture antibody. Each strip was prepared and tested with the same samples as described in (A) except the lower T line (if present) contains the CC1 capture antibody.

recombinant phytase and GM corn phytase, respectively. Therefore, serial dilutions were prepared with the recombinant phytase for the EH10a−FA7 antibody pair, with the GM phytase for the AF9a−CC1 antibody pair and tested on lateral flow strips. The detection limit to recombinant phytase using the EH10a−FA7 antibody pair was 5 ng/mL (Figure 3A), whereas the detection limit to GM phytase corn using the AF9a−CC1 antibody pair was as low as 2 ng/mL (Figure 3B). The strips were confirmed to be stable when kept at room temperature for 1 year. Reactive Antibody Binding Sites May Involve Glycosylation. As stated earlier, the EH10a−FA7 antibody pair showed a greater ability to detect the yeast-expressed phytase, whereas the AF9a−CC1 antibody pair showed a greater ability to detect the GM corn phytase. Additionally, different-sized phytase proteins could be detected from these two sources. We wanted to further explore the nature of the epitopes between yeast- and plant-expressed phytases and assess whether glycosylation has a role that determines the responses to their respective antibody match pairs. Using Western blot analysis, phytases expressed from yeast and GM corn were probed with a combined MAb mixture of EH10a, FA7, AF9a, and CC1 and monitored over time. During the first 2 weeks, the 75 kDa band dominated with a lesser abundant 60 kDa band from the yeast-expressed phytase (Figure 4A, top row). By the fourth week, this 75 kDa band was progressively less detectable, whereas the 60 kDa band was progressively more detectable (Figure 4A, top row). In contrast, the 60 kDa band from the GM corn phytase was progressively less detectable over time (Figure 4A, bottom row) and the 75 kDa was not detectable at all (data not shown). Phytase samples were prepared as above and tested over the same time periods using lateral flow strips consisting of either the EH10a−FA7 or the AF9a−CC1 antibody match pair. As

tolerance to heat are advantageous not only for feed production due to temperatures associated with pelleting but for food processing activities such as cooking and baking.6 Locations for new sources of thermotolerant as well as the engineer of technologically improved phytases to withstand increased temperatures and GM phytase crops are invaluable to industries that must deal with phytate and the challenges it represents. Characterization of Phytase Monoclonal Antibody Pairs Using Lateral Strip Assays. The additive index results indicated that the four MAbs target different phytase epitopes; therefore, lateral flow strip comparison assays using all possible MAb pair combinations were conducted and showed that the EH10a−FA7 and AF9a−CC1 MAb pairs exhibited the strongest potential to be used in lateral flow strip products (data not shown). To determine the specificity of the phytase MAb pairs, immunoassays comprising of antibody-coated lateral strips were constructed and tested with different seed varieties of commercial plants. Lateral strips that consisted of the gold-conjugated EH10a antibody and the FA7 membrane capture antibody showed that this antibody pair was able to detect the A. niger recombinant (phyA2) phytase expressed in P. pastoris and, to a lesser extent, the GM (phyA2) phytase corn (Figure 2A, lanes 1 and 2). In contrast, strips that consisted of the gold-conjugated AF9a antibody and the CC1 membrane capture antibody showed that this antibody pair was able to detect the GM phytase corn more efficiently that the recombinant phytase (Figure 2B, lanes 1 and 2). Further, phytase could not be detected in any of the test seed varieties using either of the antibody pairs, indicating a high specificity to the A. niger recombinant protein that is used in GM phytase crops. Antibody-coated lateral strips were also used to determine the sensitivity of the phytase MAb pairs. The EH10a−FA7 and AF9a−CC1 antibody pairs were able to efficiently detect the 4323

DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326

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Journal of Agricultural and Food Chemistry

Figure 3. Match pair EH10a−FA7 and AF9a−CC1 antibodies were able to detect low concentrations of phytase. (A) To determine the sensitivity of the EH10a−FA7 antibodies, concentrations from 0.002 to 200 μg/mL of recombinant phytase were applied to lateral flow strips containing the EH10a conjugate antibody and the FA7 capture antibody. (B) To determine the sensitivity of the AF9a−CC1 antibodies, concentrations from 0.001 to 400 μg/mL of the GM phytase corn were applied to lateral flow strips containing the AF9a conjugate antibody and the CC1 capture antibody. The strips were immersed in a 10 mL solution of recombinant phytase (A) and GM phytase corn (B) at the various concentrations indicated above the strips. The arrangement of the control (C) and test (T) lines along the strips is as described in Figure 2.

Figure 4. Nature of phytase epitopes affects detection by EH10a−FA7 and AF9a−CC1 match-pair MAbs over time. (A) Ten microliters of (200 μg/mL) purified recombinant phytase (phy2A) expressed from yeast (top row) and (200 μg/mL) phytase expressed from GM corn (bottom row) was prepared and evaluated every 2 weeks over a period of 10 weeks using Western blot analysis. Each blot was probed with a mixture of MAbs EH10a, FA7, AF9a, and CC1. Record dates are indicated above the blot with the protein marker sizes kilodaltons (kD) on the left. (B) Purified recombinant phytase (phy2A) expressed from yeast (top row) and phytase expressed from GM corn (bottom row) were prepared and tested along immunolateral flow strips. Test strips containing the EH10a conjugate antibody and FA7 capture antibody (green colored) and the AF9a conjugate antibody and CC1 capture antibody (brown colored) were immersed in a solution of their respectively prepared phytase, and detection was recorded on the dates indicated above the strips. The arrangement of the control (C) and test (T) lines along the strips are as described in Figure 2.

the yeast-expressed (75 kDa) phytase degrades, it becomes less detectable when using strips with the EH10a−FA7 antibody match pair (Figure 4B, top row, green strips) and becomes more detectable when using strips with the AF9a−CC1 antibody match pair over time (Figure 4B, top row, brown strips). This effect corresponds to the detection of the smallersized phytase protein, which is similar to that detected from the transgenic corn (60 kDa) phytase, which, too, becomes less detectable upon degradation using the AF9a and CC1 antibodies (Figure 4B, bottom row, brown strips) and was barely discernible on strips coated with the EH10a−FA7 antibody match pair (Figure 4B, bottom row, green strips). If the larger phytase size is indeed due to glycosylation and a loss of this glycosylation results in a change in antibody recognition, it may be tempting to assume that the epitope binding sites of one or both of the MAbs, EH10a and FA7, consists of a carbohydrate moiety. Guirakhoo et al.33 demonstrated that a carbohydrate side chain was essential in stabilizing epitopes within the envelope protein of the tick-borne encephalitis (TBE) virus. Although little is known about the structure of phytase antibody binding sites, antibodies have been shown to recognize carbohydrate epitopes.34 On the basis of our findings, one can assume that the epitope structural organization of the four MAbs is similar, albeit the carbohydrate group required for EH10a and FA7 MAbs and the antibody that prevails is dependent upon glycosylated sites and that glycosylation may be required to maintain site stability. Or one can assume that the epitopes are not similar in structure but lie near enough together so that the carbohydrate group is inhibiting or blocking the binding sites to antibodies that do not recognize

glycosylation. Cryptic sites within the TBE virus envelope containing antigenic determinants when exposed allow crossreactive antibodies to be efficiently recognized, which would not have been accessible in a native virus.35 Further studies are required such as structural analysis of the antigenic sites of the phytase protein expressed from various sources to verify the contribution of carbohydrates to antibody binding. The development of transgenic phytase plants and the call for greater awareness of the distribution of GM organisms brings about a need for accurate product verification. Further, the ability to correctly detect glycosylation within transgenic phytase plants may be beneficial when a product with a more thermostable enzyme is required. Methods to assess phytase activity usually involve detection of released inorganic phosphates by adding phytate as a substrate, requiring generation of a calibration curve and complex laboratory equipment.36−38 In this study, MAbs were generated which are able to bind to different protein epitopes that were both highly sensitive and specific to transgenic phytase, efficiently so as to be differentiated from the yeast-expressed phytase. An RDT, which is an immunochromatographic capillary flow assay consisting of MAbs, one immobilized on the surface of the membrane and the other conjugated to particles near the sample pad, allows for an efficient and sensitive point-of-care testing method that would deliver immediate results, requiring little skill or additional equipment. Here we present the 4324

DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326

Article

Journal of Agricultural and Food Chemistry

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development of such a RDT that is able to detect phytase from a GM crop.

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AUTHOR INFORMATION

Corresponding Authors

*(Jian Zheng) E-mail: [email protected]. Phone: +86 13793183297. Fax: +86 2368486780. *(Juan Zhang) Phone: +86 13793183297. Fax: +86 2368486780 Funding

This study was supported by a grant from the National Major Programs of the genetically modified organisms breeding during the 11th Five-Year Plan Period (Grant 2009ZX08012008B). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Krystyna Pangilinan for graphical assistance. ABBREVIATIONS USED MAb, monoclonal antibody; phyA2, phytase A2 gene; GM, genetically modified; AI, additive index; RDT, rapid diagnostic test

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REFERENCES

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DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326

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DOI: 10.1021/acs.jafc.5b00188 J. Agric. Food Chem. 2015, 63, 4320−4326