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, Juan Zhang, and Jian Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00188 • Publication Date (Web): 22 Apr 2015 Downloaded from http://pubs.acs.org on April 27, 2015

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Journal of Agricultural and Food Chemistry

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Development of a rapid immunochromatographic lateral flow device capable of differentiating

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phytase expressed from recombinant Aspergillus niger phyA2 and genetically modified corn

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Xiaojin Zhou†, Elizabeth Hui‡, Xiao-Lin Yu∥, Zhen Lin§, Ling-Kui Pu†, Zhiguan Tu∥, Jun

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Zhang‡,#, Qi Liu#,┴, Juan Zhang*,¥, and Jian Zheng*§

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Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12

7

†

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Zhongguancun Nandajie, Beijing 100081, PR China

9

‡

Artron BioResearch Inc, 3938 North Fraser Way, Burnaby, British Columbia, V5J 5H6, Canada

Key Laboratory of Diagnostic Medicine, Ministry of Education, Chongqing Medical University,

10

∥

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Chongqing, China. No.1 Yixueyuan Road, Yuzhong District, Chongqing 4000016, PR China

12

§

The Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education,

13

Chongqing Medical University, No.1 Yixueyuan Road, Yuzhong District, Chongqing 400016,

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PR China

15 16 17 18 19 20

#

Ji Nan Kangbo Biotechnology, 2711 Ying Xiu Road, Jinan, Shandong Province, 250101, PR

China ┴

Beijing Artron Jingbiao Biotech Inc, No. 19, Tianrong St., Daxing Bio-medicine Industry Park,

Daxing District, Beijing 102600, PR China ¥

The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical

University, No.1 Yixueyuan Road, Yuzhong District, Chongqing 400016, P.R. China

21 22 23

*

Jian Zheng; Juan Zhang, E-mail address: [email protected], Telephone: +011 86 13793183297; Fax: +86 2368486780

1 ACS Paragon Plus Environment


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ABSTRACT: Phytase is a phosphohydrolase considered highly specific for the degradation of

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phytate to release bound phosphorus for animal consumption and aid in the reduction of

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environmental nutrient loading. New sources of phytase have been sought that are economically

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and efficiently productive including the construction of genetically modified (GM) phytase

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products designed to bypass the costs associated with feed processing. Four monoclonal

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antibodies (EH10a, FA7, AF9a and CC1) raised against recombinant Aspergillus niger phyA2

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were used to develop a highly specific and sensitive immunochromatographic lateral flow device

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for rapid detection of transgenic phytase, such as in GM corn. Antibodies sequentially paired

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and tested along lateral flow strips showed that the EH10a-FA7 antibody pair was able to detect

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the recombinant yeast-phytase at 5 ng/ml whereas the AF9a-CC1 antibody pair to GM phytase

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corn at 2 ng/ml. Concurrent to this development, evidence was revealed which suggests that

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antibody binding sites may be glycosylated.

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KEYWORDS: phytase, glycosylation, monoclonal antibodies, lateral strips, GM corn


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INTRODUCTION

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Phytase (myo-inositol hexakisphosphate phosphohydrolase) EC 3.1.3.8 is part a class of

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phosphatases which can catalyze the sequential hydrolysis of phytate to lower phosphorylated

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inositol and inorganic phosphates.1,2 Phytate is the main storage form of phosphorus in livestock

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feed such as seeds and cereal grains, representing nearly 90% of their total phosphorus content.3,4

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The digestive microbial fauna of monogastric animals lack the necessary phosphorus

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hydrolyzing enzymes and as a result much undigested phytate-associated phosphorus is lost into

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the environment.5 This can lead to excessive phosphorus loading in soil and water and the

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ensuing pollution can affect other ecosystems.1,6 Phytate is considered as an antinutrient due to

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its chelating ability which results in the formation of complexes with nutritionally important

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multivalent metal ions, such as Fe3+, Zn2+, and Ca2+, making them unavailable for absorption.6,7

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Animal feed is often supplemented with inorganic phosphorus or phytase additives to meet the

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nutritional requirement for growth by increasing phosphorus availability and aid in the reduction

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in phosphorus excretion.1,8 The subsequent development and application of transgenic phytase

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plants and animals have been shown as alternatives to exogenous additives thereby reducing

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processing and formulation costs and concerns of contaminates, such as fluorine and heavy

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metals, accrued during the manufacturing process of inorganic feed phosphates.6,8,9 Higher

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levels of phytate-degrading activity are achievable by optimizing expression of specific genetic

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events and through the use of economically competitive expression/secretion systems.6,11 Such

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phytase supplementation had greatly improved the phytate antinutrient factor, prompting the

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search for more temperature and pH tolerant phytases and propelled phytase optimization

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technology through genetic and protein engineering.8,10,11 The phytase commercial market

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volume has exceeded US$250 million and is growing at around 10% per year.12



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Phytases can be glycosylated and the level of glycosylation is known to be highly

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variable.13,14,2 For example, E. coli phytase is nonglycosylated15 whereas the glycosylation

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pattern between different fungal phytases varies based on the expression system16 and between

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individuals on a given expression system.2 Glycosylation can have many effects on the

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properties of a protein, such as on stability, solubility, and metabolic energy.2 Glycosylation of

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phytase have been shown to affect thermostability13,17,18 which would be a concern regarding

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enzyme activity loss due to heat such as that from feed pelleting. Phytases for commercial use

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have been isolated mainly from fungi and bacteria6,8,11 and selection for an efficacious product is

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greatly dependent on the source1, tolerance to processing factors, digestive resistance and

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production costs.6,8,19 Major feed crops have been genetically modified to increase phytase

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expression and are at various stages of product development.20 For example, two separate

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studies on transgenic phytase barley have both reached an advanced developmental stage

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complete with field trials; however, only transgenic phytase corn (Event B23-3-1) has received

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approval status for commercial sale. Integration of such phytases as transgenes in agricultural

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products in a growing industry would benefit from inclusion of a means to verify transgenic

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phytase presence in food nutritional and environmental management strategies wherein concerns

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of phytate mineral availability and environmental issues need to be assessed. Development of a

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rapid diagnostic test (RDT), which is a qualitative immunoassay (consisting of target specific

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antibodies) used in point-of-care testing, for transgenic phytase would offer an efficient,

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convenient and affordable method of detection.

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Aspergillus niger phytase (phyA2) which has 10 potential N-glycosylation sites2 was

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cloned and expressed in a methylotrophic yeast, Pichia pastoris.21 This same phytase was

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cloned into corn, yielding seeds with a maximal phytase activity of 125 FTU/g kernels, 1000-


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fold above that of the wild type, and with 1000 g of kernels containing up to 67 times the feed

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industry requirement.22 Corn is a globally important animal feed crop; in China, corn occupies

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30 million hectares of land from which much is used to feed 500 million pigs (approximately

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50% of the global herd) and 13 billion chickens, ducks and poultry.23 As mentioned above, only

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transgenic phytase corn has been approved for commercialization, accessibility coupled with the

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ensuing demand for corn production, all make it an ideal product candidate for this project.

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Herein, two pairs of highly sensitive and specific monoclonal antibodies (MAbs) raised against

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A. niger recombinant phytase and generated through an extensive screening technique were

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described whereby each was found to detect a phytase protein of different sizes, possibly due to

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glycosylation. Using these MAbs, the transgenic corn was found to express lower levels of the

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larger-sized phytase than the recombinant phytase had expressed in yeast; however, the level of

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detection was predicated on the condition of the expressed phytase. Together, these findings

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provide important insight regarding the structural properties of phytase epitopes, suggesting a

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possible role of carbohydrates within antibody binding sites, and lead to the development of

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RDT strips coated with MAbs that can detect two different sized proteins of transgenic phytase.

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METHODS AND MATERIALS Recombinant phytase, seeds and antibodies. Aspergillus niger 963 phytase (phyA2)

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expressed in Pichia pastoris was isolated by and donated from the Biotechnology Research

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Institute, Chinese Academy of Agricultural Sciences in Beijing, China. This recombinant

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protein was freeze-dried, reconstituted in PBS (at 200 µg/ml) and stored at 4oC until required.

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This group also donated genetically modified (GM) phytase corn seeds, expressing this same A.

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niger phytase gene phyA2. Seeds from various plants [six varieties of GM corn, one variety of a



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GM soybean, corn (2733) from Pickseed, six corn varieties from West Coast Seeds (=WCS; two

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of F1, two of P, one of DF1 and one unknown), green bean, white bean and seven generic

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varieties of corn (purchased from local markets)] were used in characterization studies.

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Phytase monoclonal antibodies (MAbs = EH10a, FA7, AF9a and CC1) raised against a

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recombinant A. niger 963 (phyA2) phytase were donated by the Chinese Biotechnology Research

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Institute in Beijing and prepared in a manner similarly described in Chen et al.24

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Epitope characterization. To characterize the epitopes recognized by the purified

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phytase MAbs, an ELISA and the additivity index (AI) described by Friguet et al.25 were used.

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Briefly, wells of a 96-well plate were coated with 100 µl of 2 µg/ml recombinant phytase and

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incubated overnight at 4oC. The following day, the wells were incubated with 100 µl of

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antibodies, EH10a, FA7, AF9a, CC1, individually or in paired combinations (50 µl each) of

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equivalent concentrations at 1:1000 overnight at 4oC. The following day, the wells were

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incubated with 100 µl of a goat anti-mouse IgG-HRP secondary antibody at 1:1000 for 30 min at

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37oC. The absorbance value for each treatment was recorded at 450 nm. The AI was calculated

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using the following equation: {[2A1+2/(A1+A2)]-1}x100%, where A1, A2 and A1+2 are the

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absorbance values for the individual antibodies and the respective combined pairs. If the two

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antibodies are directed against different epitopes (no competition), A1+2 should be equal to the

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sum of A1 and A2 and the AI value should approach 100%. If the two antibodies are directed

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against the same epitope (competition), A1+2 should be equal to the mean value for A1 and A2

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and AI should be close to 0%. The threshold was determined by AI ≥ 40%.

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Protein analysis. Protein and western blot analyses on the specificity of the MAbs were

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evaluated by 15% SDS-PAGE using 1 µg of recombinant phytase and 50 mg of ground seeds

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from GM phytase corn, generic corn, green bean, white bean, Pickseed2733 corn, WCS F1 corn,


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four varieties of GM corn and one GM soybean variety. The phytase could only be released

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from the GM phytase corn by homogenizing the corn seeds in a modified Tris buffer [50 mM

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Tris-HCl (pH 8.0), 10 mM KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM β-mercaptoethanol, 0.1%

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BSA, 13% sucrose and SigmaFAST™ Protease inhibitor cocktail (Sigma-Aldrich, St. Louis,

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MO)] at 400 µl per 100 mg tissue after which the sample was centrifuged at 4500 g for 10 min.

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Laemmli loading buffer26 was then added to the supernatant and incubated at 65oC for 20 min.

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The remaining seeds were ground, homogenized in 500 µl of Laemmli buffer, boiled for 10 min,

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and centrifuged for 2 min at 12,000 g. To improved western blot detection, 5 µg of recombinant

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phytase and 100 mg of ground seeds were used. Twenty microlitres of each prepared seed

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supernatant were used for protein and western blot analysis. The recombinant phytase, 20 µl of

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water, and 10 mM Tris (the latter two as negative controls) were also boiled in Laemmli buffer

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as described above. The membrane blot was incubated overnight at 4oC with a combined

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mixture of EH10a, FA7, AF9a and CC1 MAbs (each at 1:400) whereas the SDS-PAGE protein

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gel was stained with Coomassie Brilliant Blue. The following day the membrane was incubated

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with horseradish peroxidise conjugated goat anti-mouse immunoglobulin (IgG-HRP at 1:1000)

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for 90 min at RT. Proteins on the blot were developed using the DAB (3,3'-diaminobenzidine

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tetrahydrochloride; Sigma-Aldrich, St. Louis, MO) method with 0.1% hydrogen peroxide.

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To evaluate the nature of the MAbs directed to epitopes on phytase, an additional western

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blot analysis was performed. Purified recombinant phytase protein isolated from yeast and

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ground seeds of GM phytase corn were both resuspended with PBS to a concentration of 200

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µg/ml, and stored at 4oC until required. Every 2 weeks for a period of 10 weeks, 10 µl from each

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of the recombinant phytase and GM phytase corn were boiled with 10 µl of loading buffer for 5

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min and loaded onto a 10% SDS-PAGE. Each transferred membrane blot was incubated



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overnight at 4oC with a combined mixture of MAbs as described above. The subsequent steps

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are as described above, except the concentration of the IgG-HRP secondary antibody was 1:5000

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with an incubation time of 30 min.

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Construction and evaluation along immunolateral flow test strips. The detection

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phytase antibody (the test line antibody, either FA7 or CC1) and the goat anti-mouse IgG (the

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control line antibody) were diluted to a standard concentration of 1.5 mg/ml with 10 mM Tris-

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HCl (pH 8.0) and applied in a thin line onto a nitrocellulose membrane. The colloidal gold

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conjugated phytase capture antibody was prepared as described in Chen et al.24 Briefly, 1.4 ml

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1% Na3C6H5O7 was first added to 100 ml of boiled 0.01% HAuCl4. The colloidal gold solution

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was then allowed to cool gradually after which the pH was adjusted to 8.4 with 1% K2CO3. The

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EH10a or AF9a antibodies were added dropwise into 10 ml of the colloidal gold solution and left

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to stabilize at 4oC. After 30 min, BSA blocking agent was added and the solution was placed at

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4oC for another 2 h. The mixture was centrifugated twice, the first time to recover the

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supernatant and the second the conjugate pellet which was then suspended in 10 mM borax

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buffer (with 2% BSA and 0.05% NaN3, pH 8.0). The conjugated antibody (i.e. EH10a if the

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detection antibody was FA7 or AF9a if the detection antibody was CC1) was sprayed twice onto

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a sheet of fiberglass and dried at 37oC. The phytase lateral flow test strips were assembled as

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described in Chen et al.24

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Two grams of each seed variety were ground in 10 mM Tris-HCl. The absorption pad

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end of a prepared test strip was immersed in seed supernatant. Test strips were also immersed in

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200 µg/ml of recombinant phytase, 200 µg/ml of transgenic corn, distilled water and 10 mM

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Tris-HCl (the latter two as negative controls). A response was observed within 5 min. The

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appearance of two bands, one each at the test and control site, represents a positive test result; a


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single band at the control site represents a negative test result. The absence of a line at the

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control site indicates the test is invalid.

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To determine the sensitivity of the antibodies to phytase along an immunolateral flow test

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strip, recombinant phytase was diluted in concentrations from 200 to 0.002 µg/ml by 10 mM

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Tris-HCl (pH 8.0) and tested using the EH10a-FA7 MAb match pairs whereas the transgenic

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phytase corn was tested in concentrations from 400 to 0.001 µg/ml using the AF9a-CC1 MAb

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match pairs. The responses of the strips were evaluated as above.

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The nature of the MAbs directed to epitopes on phytase was evaluated along an

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immunolateral flow test strip. Individual test strips consisting of either the EH10a-FA7 or the

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AF9a-CC1 MAb match pairs were immersed in the reconstituted recombinant or GM corn

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phytase samples every 2 weeks for a period of 10 weeks. The samples were stored at 4oC until

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required. With the “MAX” line on the test strip positioned above the liquid level, a sample was

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allowed to migrate half way up the strip after which the strip was removed. The results were

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obtained within 30 min and the strips were evaluated as above.

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The stability of test strips was assessed when kept for 1 year at RT.

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RESULTS AND DISCUSSION Monoclonal antibody characterization. Phytase is considered the most prominent

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enzyme to hydrolyze phytate and release bound phosphorus in a form available to animals for

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growth and development. A recombinant Aspergillus niger phytase (phyA2) expressed in Pichia

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pastoris21,22 was used to produce monoclonal antibodies (MAbs) employing a very extensive and

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novel screening technique.24 From a total of 976 hybridoma cell strains, 32 positive cell clones

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using repetitive ELISAs and cross-responsive screening of MAbs against A. niger (phyA2)



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phytase were generated (pers. comm., Biotechnology Research Institute). Cell lines screened for

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the strongest positive signal in phytase-challenged cultures were selected, culminating in four

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MAbs candidates (EH10a, FA7, AF9a and CC1). Additivity indices (AI) were calculated to

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determine competition for the binding sites between the four MAbs. The following AI values

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were found for the following antibody pairs: FA7+AF9a (67.7%), FA7+CC1 (91.4%),

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AF9a+EH10a (57.7%), EH10a+CC1 (69.7%), FA7+EH10a (59.6%) and AF9a+CC1 (≥99%).

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These results indicate that if any pair of these antibodies would result in the targeting of a

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different phytase epitope.

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Evaluation of monoclonal antibody pairs to phytase protein. Pichia pastoris is a

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methylotrophic yeast that when under the control of a methanol-inducible alcohol oxidase

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(AOX1) promoter has been successfully used to produce many proteins with varying degrees of

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success27,28,29 and these proteins are often glycosylated.30 Protein analyses of various seed

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varieties (Fig. 1A) were used to assess MAb specificity along with a recombinant A. niger

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phytase expressed from yeast and a genetically modified (GM) corn containing this same A.

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niger phytase. The samples were probed with a MAb mixture of EH10a, FA7, AF9a and CC1

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(in equal proportions). The recombinant A. niger phytase expressed from yeast shows a band of

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approximately 75 kD (Fig. 1B, lane 4) whereas the GM A. niger phytase corn reveals a band of

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about 60 kD (Fig. 1B, lane 5). Further, this MAb mixture did not cross-react with the other test

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seeds as shown from western blot analysis (Fig. 1B, lanes 6-15). The larger sized protein from

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the yeast-expressed phytase may be due to the greater degree of glycosylation often observed in

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proteins expressed from yeast. Differences in glycosylation pattern expressed from different

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hosts may be attributed to the discrete processes found in their respective cellular environments.

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For example, A. fumigates was found to produce a phytase that can tolerate boiling at 100oC for


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20 min, retaining 90% of its initial activity.31 Lucca et al.32 found that when rice containing the

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A. fumigates phytase was boiled for 20 min, the enzyme had retained 50% of the enzyme

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activity. However, when rice seeds were cooked under the same conditions, only 8% of the

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phytase activity was retained. Similarly, these differences in thermo-tolerance may be attributed

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to the different glycosylation patterns in phytase expressed between diverse hosts. Phytases

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which have high tolerance to heat are advantageous not only for feed production due to

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temperatures associated with pelleting but for food processing activities such as cooking and

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baking.6 Locations for new sources of thermo-tolerant as well as the engineer of technologically

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improved phytases to increased temperatures and GM phytase crops are invaluable to industries

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that must deal with phytate and the challenges it represents.

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Characterization of phytase monoclonal antibody pairs using lateral strip assays.

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The additive index results indicated that the four MAbs target different phytase epitopes;

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therefore, lateral flow strip comparison assays using all possible MAb pair combinations were

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conducted and showed that the EH10a-FA7 and AF9a-CC1 MAb pairs exhibited the strongest

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potential to be used in lateral flow strip products (data not shown). To determine the specificity

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of the phytase MAb pairs, immunoassays comprising of antibody-coated lateral strips were

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constructed and tested with different seed varieties of commercial plants. Lateral strips which

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consisted of the gold-conjugated EH10a antibody and the FA7 membrane capture antibody

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showed that this antibody pair was able to detect the A. niger recombinant (phyA2) phytase

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expressed in P. pastoris and, to a lesser extent, the GM (phyA2) phytase corn (Fig. 2A; lanes 1

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and 2). In contrast, strips which consisted of the gold-conjugated AF9a antibody and the CC1

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membrane capture antibody showed that this antibody pair was able to detect the GM phytase

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corn more efficiently that the recombinant phytase (Fig. 2B; lanes 1 and 2). Further, phytase



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could not be detected in any of the test seed varieties using either of the antibody pairs,

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indicating a high specificity to the A. niger recombinant protein which is used in GM phytase

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crops.

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Antibody-coated lateral strips were also used to determine the sensitivity of the phytase

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MAb pairs. The EH10a-FA7 and AF9a-CC1 antibody pairs were able to efficiently detect the

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recombinant phytase and GM corn phytase, respectively. Therefore, serial dilutions were

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prepared with the recombinant phytase for the EH10a-FA7 antibody pair, with the GM phytase

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for the AF9a-CC1 antibody pair and tested on lateral flow strips. The detection limit to

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recombinant phytase using the EH10a-FA7 antibody pair was 5 ng/ml (Fig. 3A) whereas the

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detection limit to GM phytase corn using the AF9a-CC1 antibody pair was as low as 2 ng/ml

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(Fig. 3B). The strips were confirmed to be stable when kept at RT for 1 year.

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Reactive antibody binding sites may involve glycosylation. As stated earlier, the

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EH10a-FA7 antibody pair showed a greater ability to detect the yeast-expressed phytase whereas

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the AF9a-CC1 antibody pair showed a greater ability to detect the GM corn phytase.

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Additionally, different sized phytase proteins could be detected from these two sources. We

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wanted to further explore the nature of the epitopes between yeast- and plant-expressed phytases

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and assess whether glycosylation has a role that determines the responses to their respective

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antibody match pairs. Using western blot analysis, phytases expressed from yeast and GM corn

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were probed with a combined MAb mixture of EH10a, FA7, AF9a and CC1 and monitored over

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time. During the first two weeks, the 75 kD band dominated with a lesser abundant 60 kD band

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from the yeast-expressed phytase (Fig. 4A, top row). By the fourth week, this 75 kD band was

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progressively less detectable whereas the 60 kD band was progressively more detectable (Fig.

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4A, top row). In contrast, the 60 kD band from the GM corn phytase was progressively less


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detectable over time (Fig. 4A, bottom row) and the 75 kD was not detectable at all (data not

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shown).

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Phytase samples were prepared as above and tested over the same time periods using

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lateral flow strips consisting of either the EH10a-FA7 or the AF9a-CC1 antibody match pair. As

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the yeast-expressed (75 kD) phytase degrades, it becomes less detectable when using strips with

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the EH10a-FA7 antibody match pair (Fig. 4B, top row, green strips) and becomes more

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detectable when using strips with the AF9a-CC1 antibody match pair over time (Fig. 4B, top

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row, brown strips). This effect corresponds to the detection of the smaller sized phytase protein

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which is similar to that detected from the transgenic corn (60 kD) phytase which too, becomes

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less detectable upon degradation using the AF9a and CC1 antibodies (Fig. 4B, bottom row,

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brown strips) and was barely discernible along strips coated with the EH10a-FA7 antibody

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match pair (Fig. 4B, bottom row, green strips). If the larger phytase size is indeed due to

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glycosylation and a loss of this glycosylation results in a change in antibody recognition, it may

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be tempting to assume that the epitope binding sites of one or both of the MAbs, EH10a and

282

FA7, consists of a carbohydrate moiety. Guirakhoo et al.33 demonstrated that a carbohydrate

283

side chain was essential in stabilizing epitopes within the envelope protein of the tick-borne

284

encephalitis (TBE) virus. Although little is known about the structure of phytase antibody

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binding sites, antibodies have been shown to recognize carbohydrate epitopes.34 Based on our

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findings, one can assume that the epitope structural organization of the four MAbs is similar,

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albeit the carbohydrate group required for EH10a and FA7 MAbs, and the antibody that prevails

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is dependent upon glycosylated sites and that glycosylation may be required to maintain site

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stability. Or one can assume that the epitopes are not similar in structure but lie near enough

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together in which the carbohydrate group is inhibiting or blocking the binding sites to antibodies



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that do not recognize glycosylation. Cryptic sites within the TBE virus envelope containing

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antigenic determinants when exposed allow cross-reactive antibodies to be efficiently recognized

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which would not have been accessible in a native virus.35 Further studies are required such as

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structural analysis of the antigenic sites of the phytase protein expressed from various sources to

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verify the contribution of carbohydrates to antibody binding.

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The development of transgenic phytase plants and the call for greater awareness of the

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distribution of GM organisms brings about a need for accurate product verification. Further, the

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ability to correctly detect glycosylation within transgenic phytase plants may be beneficial when

299

a product with a more thermostable enzyme is required. Methods to assess phytase activity

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usually involve detection of released inorganic phosphates by adding phytate as a substrate,

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requiring generation of a calibration curve and complex laboratory equipment.36,37,38 In this

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study, MAbs were generated which are able to bind to different protein epitopes that were both

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highly sensitive and specific to transgenic phytase; efficiently so as to be differentiated from the

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yeast-expressed phytase. A rapid diagnostic test (RDT) which is an immunochromatographic

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capillary flow assay consisting of MAbs, one immobilized on the surface of the membrane and

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the other conjugated to particles near the sample pad, allows for an efficient and sensitive point-

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of-care testing method that would deliver immediate results, requiring little skill or additional

308

equipment. Here we present the development of such a RDT that is able to detect phytase from a

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GM crop.

310 311

ABBREVIATIONS: MAb, monoclonal antibody; phyA2, phytase A2 gene; GM, genetically

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modified; AI, additive index; RDT, rapid diagnostic test.

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ACKNOWLEDGEMENTS. This study was supported by a grant from the National Major

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Programs of the genetically modified organisms breeding during the 11th Five-Year Plan Period

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(Grant No. 2009ZX08012-008B). We thank Krystyna Pangilinan for graphical assistance.

317 318

Note: The authors declare no financial conflicts of interest.

319



320

REFERENCES

321

(1) Bohn, L.; Meyer, A. S.; Rasmussen, S. K. Phytate: impact on environment and human

322

nutrition. A challenge for molecular breeding. J. Zhejiang Univer. Sci. B 2008, 9, 165-191.

323

(2) Wyss, M.; Pasamontes, L.; Friedlein, A.; Rémy, R.; Tessier, M.; Kronenberger, A.;

324

Middendorf, A.; Lehmann, M.; Schnoebelen, L.; Röthlisberger, U.; Kusznir, E.; Wahl, G.;

325

Müller, F.; Lahm, H.-W.; Vogel, K.; van Loon, A. P. G. M. Biophysical characterization of

326

fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size,

327

glycosylation pattern, and engineering of proteolytic resistance. Appl. Environ. Microbiol. 1999,

328

65, 359-366.

329

(3) Kikunaga, S.; Takahashi, M.; Huzisige, H. Accurate and simple measurement of phytic acid

330

contents in cereal grains. Plant Cell Physiol. 1985, 26, 1323-1330.

331

(4) Reddy, N. R.; Sathe, S. K.; Salunkhe, D. K. Phytates in legumes and cereals. Adv. Food Res.

332

1982, 28, 1-92.

333

(5) Ravindran, V.; Bryden, W. L.; Kornegay, E. T. Phytates: occurrence, bioavailability and

334

implications in poultry nutrition. Poult. Avian Biol. Rev. 1995, 6, 125-43.

335

(6) Greiner, R.; Konietzny, U. Phytase for food application. Food Technol. Biotechnol. 2006,

336

44, 125-140.

337

(7) Lopez, H. W.; Leenhardt, F.; Coudray, C.; Remesy, C. Minerals and phytic acid interactions:

338

is it a real problem for human nutrition? Int. J. Food. Sci. Technol. 2002, 37, 727–739.

339

(8) Haefner, S.; Knietsch, A.; Scholten, E.; Braun, J.; Lohscheidt, M.; Zelder, O.

340

Biotechnological production and applications of phytases. Appl. Microbiol. Biotechnol. 2005,

341

68, 588-597. 16 ACS Paragon Plus Environment

Page 16 of 28

Page 17 of 28


342

(9) Hoffmann, J.; Hoffmann, K.; Skut, J.; Huculak-Maczka, M. Modification of manufacturing

343

process of feed phosphates. CHEMIK 2011, 3, 194-191.

344

(10) Lei, X. G.; Weaver, J. D.; Mullaney, E.; Ullah, A. H.; Azain, M. J. Phytase, a new life for

345

an“old” enzyme. Annu. Rev. Anim. Biosci. 2013, 1, 283–309.

346

(11) Yao, M.-Z.; Zhang, Y.-H.; Lu, W.-L.; Hu, M.-Q.; Wang, W.; Liang, A.-H. Phytases:

347

crystal structures, protein engineering and potential biotechnological applications. J. Appl.

348

Microbiol. 2011, 112, 1-14.

349

(12) Greiner, R. “Update on Characteristics of Commercial Phytases”, Paper presented at the

350

International Phytase Symposium 2012, Dec. 11-13, Rome, Italy.

351

(13) Han, Y.; Lei, X. G. Role of glycosylation in the functional expression of an Aspergillus

352

niger phytase (phyA) in Pichia pastoris. Arch. Biochem. Biophy. 1999, 364, 83–90.

353

(14) Panchal, T.; Wodzinski, R. J. Comparison of glycosylation patterns of phytase from

354

Aspergillus niger (A. ficuum) NRRL 3135and recombinant phytase. Prep. Biochem. Biotechnol.

355

1998, 28, 201-217.

356

(15) Phillippy, B. Q.; Mullaney, E. J. Expression of an Aspergillus niger phytase (phyA) in

357

Escherichia coli. J. Agric. Food. Chem. 1997, 45, 3337-3342.

358

(16) Ullah, A. H. J.; Sethumadhavan, K.; Mullaney, E. J.; Ziegelhoffer, T.; Austin-Phillips, S.

359

Fungal phyA gene expressed in potato leaves produces active and stable phytase. Biochem.

360

Biophys. Res. Comm. 2003, 306, 603–609.

361

(17) Guo, M.; Hang, H.; Zhu, T.; Zhuang, Y.; Chu, J.; Zhang, S. Effect of glycosylation on

362

biochemical characterization of recombinant phytase expressed in Pichia pastori. Enz.

363

Microbial. Technol. 2008, 42, 340–345. 17 ACS Paragon Plus Environment


364

(18) Han, Y.; Wilson, D. B.; Lei, X. G. Expression of an Aspergillus niger phytase gene (phyA)

365

in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 1999, 65, 1915-1918.

366

(19) Lei, X. G.; Stahl, C. H. Biotechnological development of effective phytases for mineral

367

nutrition and environmental protection. Appl. Microbiol. Biotechnol. 2001, 57, 474–481.

368

(20) Tillie, P.; Dillen, K.; Rodriguez-Cerezo, E. The Pipeline of GM Crops for Improved

369

Animal Feed. In: Animal Nutrition with Transgenic Plants; Flachowsky, G., Ed.; CABI

370

Biotechnology Series; Wallingford, Oxfordshire, UK, 2013, 231pp.

371

(21) Yao, B.; Zhang, C.; Wang, J.; Fan Y. Recombinant Pichia pastoris overexpressing

372

bioactive phytase. Sci. China (Ser C) 1998, 41, 330-336.

373

(22) Chen, R.; Zhang, C.; Yao, B.; Xue, G.; Yang, W.; Zhou, X.; Zhang, J.; Sun, C.; Chen, P.;

374

Fan, Y. Corn seeds as bioreactors for the production of phytase in the feed industry. J.

375

Biotechnol. 2013, 165, 120-126.

376

(23) ISAAA; Brief 41-2009: Executive Summary ISAAA, 2009. Global Status of

377

commercialized Biotech/GM Crops: 2009. The first fourteen years, 1996 to 2009.

378

(http://isaaa.org/resources/publications/briefs/41/executivesummary/) (Last accessed 2015).

379

(24) Chen, W.; Zhang, J.; Lu, G.; Yuan, Z.; Wu, Q.; Li, J.; Xu, G.; He, A.; Zheng, J.; Zhang, J.

380

Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio

381

cholera 01 serotype Ogawa. Clin. Biochem. 2014, 47, 448-454.

382

(25) Friguet, B.; Djavadi-Ohaniance, L.; Pages, J.; Bussard, A.; Goldberg, M. A convenient

383

enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the

384

same antigenic site. Application to hybridomas specific for the β2-subunit of Escherichia coli

385

tryptophan synthase. J. Immunol. Methods 1983, 60, 351-358.


Page 18 of 28

Page 19 of 28


386

(26) Laemmli, U. K. Cleavage of Structural Proteins during the Assembly of the Head of

387

Bacteriophage T4. Nature 1970, 227, 680-685.

388

(27) Xuan, N. T.; Hang, M. T.; Thanh, V. N. Cloning and over expression of an Aspergillus

389

niger XP phytase gene (phyA) in Pichia pastoris. World Acad. Sci. Eng. Technol. 2009, 56,

390

750-753.

391

(28) Xiong, A. S.; Yao, Q. H.; Peng, R. H.; Han, P. L.; Cheng, Z. M.; Li, Y. High level

392

expression of a recombinant acid phytase gene in Pichia pastoris. J. Appl. Microbiol. 2005, 98,

393

418-28.

394

(29) Sreekrishna, K.; Brankamp, R. G.; Kropp, K. E.; Blankenship, D. T.; Tsay, J.-T.; Smith, P.

395

L.; Wierschke, J. D.; Subramaniam, A.; Birkenberger, L. A. Strategies for optimal synthesis and

396

secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 1997, 190,

397

55-62.

398

(30) Casey, A.; Walsh, G. Identification and characterization of a phytase of potential

399

commercial interest. J. Biotechnol. 2004, 110, 313–322.

400

(31) Pasamontes, L.; Haiker, M.; Wyss, M.; Tessier, M.; van Loon, A. P. Gene cloning,

401

purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigates.

402

Appl. Environ. Microbiol. 1997, 63, 1696-700.

403

(32) Lucca, P.; Hurrell, R.; Potrykus, I. Genetic engineering approaches to improve the

404

bioavailability and the level of iron in rice grains. Theor. Appl. Genet. 2001, 102, 392–397.

405

(33) Guirakhoo, F.; Heinz, F. X.; Kunz, C. Epitope model of tick-borne encephalitis virus

406

envelope glycoprotein E: Analysis of structural properties, role of carbohydrate side chain, and

407

conformational changes occurring at acidic pH. Virology 1989, 169, 90-99. 19 ACS Paragon Plus Environment


408

(34) Plum, M.; Michel, Y.; Wallach, K.; Raiber, T.; Blank, S.; Bantleon, F. I.; Diethers, A;

409

Greunke, K.; Braren, I.; Hackl, T.; Meyer, B.; Spillner, E. Close-up of the immunogenic α 1,3-

410

galactose epitope as defined by a monoclonal chimeric immunoglobulin E and human serum

411

using saturation transfer difference (STD) NMR. J. Biol. Chem. 2011, 286, 43103-43111.

412

(35) Stiasny, K.; Kiermayr, S.; Holzmann, H.; Heinz, F. X. Cryptic properties of a cluster of

413

dominant Flavivirus cross-reactive antigenic sites. J. Virol. 2006, 80, 9557-9568.

414

(36) Abd-EIAziem, F.; Greiner, R. Recombinant bacterial phytases to reduce environmental

415

phosphate pollution. In Environmental Biotechnology: Advancement in Water and Wastewater

416

Application in the Tropics; Ujang, Z.; Henze, M., Eds.; IWA Publishing: London, UK, 2004; pp

417

165-172.

418

(37) Berry, D. F.; Harich, K. Tetrachlorofluorescein TInsP5 as a substrate analog probe for

419

measuring phytase activity in surface water: Proof of concept. J. Environ. Qual. 2012, 42, 56-

420

64.

421

(38) Heinonen, J.K.; Lahti, R.J. A new and convenient colorimetric determination of inorganic

422

orthophosphate and its application to the assay of inorganic phosphates. Anal. Biochem. 1981,

423

113, 313-317.

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425

LEGENDS

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Figure 1. Monoclonal antibodies (MAbs) target phytase. (A) Fifty micrograms of plant seeds

427

were ground in 500 µl Laemmli buffer of which 20 µl were loaded into each lane. The lanes are

428

as follows: (1) water, (2) 10 mM Tris-HCl, pH 8.0, (3) Marker, (4) 1 µg recombinant phytase,

429

(5) GM phytase corn, (6) corn 1, (7) green bean, (8) white bean, (9) Pickseed2733 corn, (10)

430

WCS F1 corn, (11) GM corn 1 , (12) GM corn 2, (13) GM corn 3, (14) GM corn 4 and (15) GM

431

soybean. (B) Western blot analyses were conducted on the samples used in (A), except that 5 µg

432

of recombinant phytase and 100 mg of ground plant seeds were used and probed with an equal

433

mixture of MAbs EH10a, FA7, AF9a and CC1. The protein marker sizes kilodaltons (kD) are

434

indicated on the left of the blot. GM = genetically modified; WCS = West Coast Seeds. Unless

435

stipulated, GM corn samples are not GM phytase corn. Additional methods are described in the

436

text.

437 438

Figure 2. Both match pair EH10a-FA7 and AF9a-CC1antibodies show high specificity to

439

phytase. (A) Phytase specificity analysis was conducted using lateral flow strips containing the

440

EH10a conjugate antibody and the FA7 capture antibody. Strips 1 – 4 were immersed in a 10 ml

441

solution consisting of (1) 200 µg/ml recombinant phytase, (2) 200 µg/ml transgenic phytase corn,

442

(3) 10 mM Tris-HCl and (4) distilled water. Strips 5 – 28 were immersed in the supernatant of 2

443

g of seeds ground in 10 ml of 10 mM Tris-HCl for 1-5 minutes. The strips were tested using the

444

following seeds: (5) corn 1, (6) corn 2, (7) corn 3, (8) corn 4, (9) GM corn 1, (10) GM soybean,

445

(11) Pickseed2733 corn, (12) GM corn 2, (13) GM corn 3, (14) GM corn 4, (15) WCS F1 corn 1,

446

(16) WCS F1 corn 2, (17) WCS P corn 1, (18) WCS P corn 2, (19) WCS DF1 corn, (20) green

447

soybean, (21) WCS corn, (22) green bean, (23) white bean, (24) corn 5, (25) corn 6, (26) corn 7,



448

(27) GM corn 5 and (28) GM corn 6. For each strip the upper line is the control (C) line

449

containing antibodies to goat anti-mouse IgG and the lower line (if present) is the test (T) line

450

with the FA7 capture antibody. The corn samples used in strips 5 to 8 and 24 to 26 were generic

451

(non-GM) varieties. (B) Phytase specificity analysis was conducted using lateral flow strips

452

containing the AF9a conjugate antibody and the CC1 capture antibody. Each strip was prepared

453

and tested with the same samples as described in Figure 2(a) except the lower T line (if present)

454

contains the CC1 capture antibody.

455 456

Figure 3. Match pairs EH10a-FA7 and AF9a-CC1 antibodies were able to detect low

457

concentrations of phytase. (A) To determine the sensitivity of the EH10a-FA7 antibodies,

458

concentrations from 0.002 to 200 µg/ml of recombinant phytase were applied to lateral flow

459

strips containing the EH10a conjugate antibody and the FA7 capture antibody. (B) To determine

460

the sensitivity of the AF9a-CC1 antibodies, concentrations from 0.001 to 400 µg/ml of the GM

461

phytase corn were applied to lateral flow strips containing the AF9a conjugate antibody and the

462

CC1 capture antibody. The strips were immersed in a 10 ml solution of recombinant phytase (A)

463

and GM phytase corn (B) in the various concentrations indicated above the strips. The

464

arrangement of the control (C) and test (T) lines along the strips is as described in Figure 2.

465 466

Figure 4. The nature of phytase epitopes affects detection by EH10a-FA7 and AF9a-CC1 match-

467

pair MAbs over time. (A) Ten microlitres of (200 µg/ml) purified recombinant phytase (phy2A)

468

expressed from yeast (top row) and (200 µg/ml) phytase expressed from GM corn (bottom row)

469

were prepared and evaluated every 2 weeks over a period of 10 weeks using western blot

470

analysis. Each blot was probed with a mixture of MAbs EH10a, FA7, AF9a and CC1. Record


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471

dates are indicated above the blot with the protein marker sizes kilodaltons (kD) on the left. (B)

472

Purified recombinant phytase (phy2A) expressed from yeast (top row) and phytase expressed

473

from GM corn (bottom row) were prepared and tested along immunolateral flow strips. Test

474

strips containing the EH10a conjugate antibody and the FA7 capture antibody (green coloured)

475

and AF9a conjugate antibody and the CC1 capture antibody (brown coloured) were immersed in

476

a solution of their respectively prepared phytase and detection was recorded on the dates

477

indicated above the strips. The arrangement of the control (C) and test (T) lines along the strips

478

are as described in Figure 2.

479



480

For Table of Contents Only

481


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Page 25 of 28


(A) kD

1

2 3 4 5

6 7 8 9 10 11 12 13 14 15

95 > 70 > 42 >

(B) kD

1 2 3 4

5 6 7 8 9 10 11 12 13 14 15

95 > 70> 42 >

Figure 1 ACS Paragon Plus Environment


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(A) 1 2

3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

C line> T line>

(B)

1 2

3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

C line> T line>


Page 27 of 28


(A) μg/ml 200

20 10

2

1

0.1 .05 .02 .01 .005 .002

μg/ml 400 200 20 10

1

.1 .05 .01 .02 .005 .002 .001

C line > T line >

(B)

C line> T line>



Page 28 of 28

(A) 0

2

4

6

8

10

Time (wk) 0

2

4

6

8

10

Time (wk) 75 kD> 60 kD> 60 kD>

(B)

C line> T line >

C line> T line>

EH10a-FA7 matched pair strip AF9a-CC1 matched pair strip


Development of a Rapid Immunochromatographic Lateral Flow Device

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