DEVELOPMENT OF MONOCLONAL ANTIBODY BASED IMMUNOASSAYS FOR TREE NUT DETECTION AND QUANTIFICATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0207219
• Grant No.: 2006-35503-17229
• Proposal No.: 2008-05271
• Project Start Date: Sep 1, 2006
• Project End Date: Aug 31, 2010
• Grant Year: 2009
• Program Code: [71.1]- Improving Food Quality and Value

Recipient Organization
FLORIDA STATE UNIVERSITY
118 N. WOODWARD AVE
TALLAHASSEE,FL 32306

Performing Department
(N/A)

Non Technical Summary
Currently there are no methods available that: 1) can help validate available assays for tree nut detection and quantification and 2) can adequately address the issue of antibody (Ab) cross-reactivity that may lead to false positive identification of the offending food. We propose to develop monoclonal antibody (MAb)-based immunoassays to 1) detect and quantify trace amounts of almonds and cashew nuts, in various foods, as both are often implicated in tree nut-induced allergies in humans; and 2) assess effects of various processing methods on the stability of select epitopes on targeted almond and cashew nut proteins The selection of nut seed proteins will be based on their demonstrated stability against several processing methods commonly encountered in processing the tree nuts, either alone or as an ingredient, in a variety of food uses. Results of experiments directed at assessing effects of food processing on epitope stability will, for the first time, define the stability of epitopes on specific allergenic protein(s) in almonds and cashew nuts leading to improved fundamental understanding of epitope stability thereby helping in the design of specific processing methods to inactivate defined epitopes, thus improving the safety of consumers specifically sensitive to those epitopes. The information gained will provide critical information to help improve food labeling and consumer food safety.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1219	1090	10%
503	1219	1040	10%
504	1219	1080	10%
711	1212	1000	70%

Knowledge Area
206 - Basic Plant Biology; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 504 - Home and Commercial Food Service; 503 - Quality Maintenance in Storing and Marketing Food Products;

Subject Of Investigation
1212 - Almond; 1219 - Edible tree nuts, general/other;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1000 - Biochemistry and biophysics; 1080 - Genetics;

Keywords

monoclonal antibody

tree nut

elisa

food allergen

Goals / Objectives
The objectives of the proposed research are to develop specific, sensitive (less than or equal to 10 parts per million), and robust, immunoassays for detection and quantification of almonds and cashew nuts in foods and food products. Additionally, the effects of a wide range of food matrices, food processing methods, and food storage conditions on the optimized assays will be investigated to assess suitability of the assays for practical use.

Project Methods
The overall approach is to use molecularly defined reagents such as mouse monoclonal antibodies (MAbs) and, where possible, cloned target antigens. Two formats of enzyme-linked immunosorbent assays (ELISAs), inhibition and capture, will be assessed. For assay development, MAbs will be selected that are devoid of cross-reactivity and are directed against stable epitopes that are not compromised by food processing. The proteins used for MAb production will contain 1:2 (w/w) mixture of proteins extracted from unprocessed and processed nut seeds. The processed component will include equal amounts of roasted, blanched, fried, autoclaved, and microwaved tree nuts. For assessment of the effects of processing and storage, the stable epitopes will be compared to MAb-epitope combinations that are labile. Where possible recombinant indicator proteins, rather than their heterogeneous and less well-defined native nut seed-derived counterparts, will be used as defined components of the assays. To assess the effects of food matrices, selected high affinity MAbs will be tested for interference by variety of foods that have been spiked with known quantities of the target nut protein. This testing will be done in three phases. In the first, ground foods will be spiked with known amounts of target nut extracts and tested. In the second, ground foods will be spiked with target nut flours and co-extracted, and in the third, foods will be processed (e.g. baked, cooked, stir fried) to include known amounts of target nut ingredients and their extracts tested by ELISA.

Progress 09/01/06 to 08/31/10

Outputs
OUTPUTS: Mouse monoclonal antibody-based enzyme linked immunosorbent assays (ELISAs) for specific, sensitive (part per million- ppm), and robust detection and quantification of trace amounts of lmond and cashew nuts were developed. Specifically, we have identified 11S legumin amandin (Pru du 6.01) and 11S legumin anacardein (Ana o 2) as an excellent, stable markers for the purpose of detecting trace amounts of almond and cashew nut seeds, respectively. During the project period, we developed mouse monoclonal antibodies (mAbs) targeted against Pru du 6.01 and Ana o 2 and using these mAbs as detecting agents, we developed sensitive (detection sensitivity of 20-50 ng/ml with a detection range of 10-300 ng/ml), specific, and robust ELISAs. Using these mouse mAb-based ELISAs we also demonstrated the stability of Pru du 6.01 and Ana o 2 towards common food processing treatments including autoclaving, blanching, gamma-irradiation, microwave heating, roasting (dry as well as oil), frying, and pH (range 1-13). Using the mouse mAbs directed against two additional allergenic proteins in cashew nut seeds- a vicilin 7S protein (Ana o 1) and a 2S protein (Ana o 3) proteins we developed additional mAb-based ELISAs (each with a of sensitivity 20-50 ng/ml). Using these mAb-based ELISAs we also demonstrated the stability of Ana o 1, and Ana o 3 towards common food processing treatments. In related projects, almond, pistachio, and pecan cDNA libraries have been constructed and a Brazil nut cDNA library has been procured to identify and characterize additional allergens in the corresponding seeds. We have demonstrated the stability of allergenic proteins in pecan and Brazil nut seeds. Studies have been initiated to identify and characterize epitopes recognized by human IgE and mouse mAbs. The research results have been disseminated at several national and international meetings, USDA PD workshops, and through scientific publications in refereed journals and invited book chapters/reviews. PARTICIPANTS: Individual who worked on the project Shridhar K. Sathe, PI, Department of Nutrition, Food & Exercise Sciences, Florida State University, Oversaw all projects, assisted with project design, data interpretation and manuscript writing. Kenneth H. Roux, co-PI, Department of Biological Sciences, Flordia State University, Contributed to project management, experimental design and manuscript writing. Suzanne S. Teuber, Clinical Collaborator, Department of Internal Medicine, Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, School of Medicine, Davis, CA Tawde, Pallavi, Graduate student, Department of Biological Sciences, Florida State University, Performed and completed her doctoral research project. Harshal Kshirsagar, Graduate student, Department of Nutrition, Food & Exercise Sciences, Florida State University, Performed and completed his doctoral research project. Girdhari Sharma, Graduate student, Department of Nutrition, Food & Exercise Sciences, Florida State University, Performed and completed his doctoral research project. Erin Monaghan, Graduate student, Department of Nutrition, Food & Exercise Sciences, Florida State University, Performed and completed her doctoral research project. Mengna Su, Graduate student, Department of Nutrition, Food & Exercise Sciences, Florida State University, Conducting her doctoral research project. LeAnna Willison, Graduate student, Department of Biological Sciences, Florida State university, Performed and completed her research for her masters research project. She is currently conducting her doctoral research project. Shyamali Jayasena, Graduate student, Department of Nutrition, Food & Exercise Sciences, Florida State University, Conducting her masters research project. Aditya Joshi, Graduate student, Department of Nutrition, Food & Exercise Sciences, Florida State University, Conducting his masters research project. Partnering organizations: Department of Internal Medicine, Division of Rheumatology, Allergy and Clinical Immunology, University of California Davis, School of Medicine, Davis, CA Collaborators and contacts: Thomas E. Thompson, PhD, USDA-ARS Pecan Breeding and Genetics, Sommerville, TX Hugh Sampson, MD, Professor of Pediatrics, Dean for Translational Biomedical Sciences, Director, Jaffe Food Allergy Institute, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY Martin Wickham, PhD, Institute of Food Research (IFR), Norwich, United Kingdom Clare Mills, PhD, Institute of Food Research (IFR), Norwich, United Kingdom EURO-PREVALL, A European Consortium for Food Allergy Research Training or professional development: Eleven graduate students, three undergraduate students, and two postdocs have worked on this project. In all cases, the persons were involved in the research aspect of the project. TARGET AUDIENCES: Food processing industry, regulatory agencies, and the consumer interested in almond and cashew allergies. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Mouse monoclonal antibody-based enzyme linked immunosorbent assays (ELISAs) for specific, sensitive (part per million- ppm), and robust detection and quantification of trace amounts of almond and cashew nuts were developed. The resources not only helped develop mAb-based assays for almond and cashew nut detection, they also helped further our understanding of several additional allergens in many tree nuts including Brazil nut, pecan, and pistachio. A new method for simultaneous isolation and purification of three native allergenic proteins from Brazil nut seeds was developed. A new rabbit polyclonal antibody based sensitive, specific, and robust ELISA was also developed to detect ng quantities of Brazil nut proteins. Additionally, three allergenic pecan proteins (an 11S legumin, a 7S vicilin, and a 2S albumin) were identified, sequenced and two (the legumin and the 2S albumin) were further characterized for their epitopes protein motifs recognized by the pecan allergic patient IgE). The project research has helped generate almond, pistachio, and pecan cDNA libraries, identification and characterization additional allergenic proteins in several tree nuts, and foster collaborations and contacts with potential for further research in improving our understanding of tree nut allergies.

Publications

• Refereed 1. Venkatachalam, M., Teuber, S. S., Peterson, W. R., Roux, K. H., and Sathe, S. K. 2006. Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch]: Effects of thermal treatments and in vitro digestion. J. Agric. Food Chem. 54(4):1449-1458.
• 2. Venkatachalam, M., and Sathe, S. K. 2006. Chemical composition of selected edible nuts. J. Agric. Food Chem. 54(13):4705-4714.
• 3. Tawde, P., Venkatesh, Y. P., Wang, F., Teuber, S. S., Sathe, S. K., and Roux, K. H. 2006. Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen. J. Allergy Clin Immunol. J. Allergy Clin. Immunol.118:915-922.
• 4. Venkatachalam, M., Thompson, T. E., Khsirsagar, H. H., Roux, K. H., and Sathe, S. K. 2007. Biochemical composition and immunological comparison of select pecan [Caya illinoinensis (Wangenh.) K. Koch] cultivars. J. Agric. Food Chem.55:9899-9907.
• 5. Monaghan, E. K., Venkatachalam, M., Seavy, M., Beyer, K., Sampson, H. A., Roux, K. H., and Sathe, S. K. 2008. Enzyme linked immunosorbent assay (ELISA) for detection of sulfur-rich protein (SRP) in soybeans (Glycine max L.) and certain other edible plant seeds. J. Agric. Food Chem. J. Agric. Food Chem. 56:765-777.
• 6. Venkatachalam, M., Roux, K. H., and Sathe, S. K. 2008. Biochemical characterization of soluble proteins in pecan [Carya illinoinensis (Wangenh.) K. Koch]. J. Agric. Food Chem. 56:8103-8110.
• 7. Comstock, S., Robotham, J. M., Tawde, P., Kshirsagar, H. H., Sathe, S. K., Roux, K. H., and Teuber, S. S. 2008. Immunoglobulin E-reactive proteins in cashew (Anacardium occidentale) apple juice concentrate. J. Agric. Food Chem. 56:5977-5982.
• 8. Willison, L. Tawde, P., Robotham, J., Penny, R., Teuber, S. S., Sathe, S. K., and Roux, K. H. 2008. Pistachio vicilin, Pis v 3, is IgE-reactive and cross-reacts with the homologous cashew allergen, Ana o 1. Clin. Exptl. Allergy. 38:1229-1238.
• 9. Venkatachalam, M., Monaghan, E. K., Kshirsagar, H. H., Robotham, J. M., O'Donnell, S. E., Gerber, M. S., Roux, K. H., and Sathe, S. K. 2008. Effects of processing on immunoreactivity of cashew nut (Anacardium occidentale L.) seed flour proteins. J. Agric. Food Chem. 56(19):8998-9005.
• 10. Sathe, S. K., Seeram, N. P., Kshirsagar, H. H., Heber, D., and Lapsley, K. A. 2008. Fatty acid composition of California grown almonds. J. Food Sci. 73:C607-C614.
• 11. Sathe, S. K. and Sharma, G. M. 2009. Effects of food processing on food allergens. Mol. Nutr. Food Res. 53:970-978.
• 12. Sathe, S. K., Sharma, G. M., Kshirsagar, H. H., Su, M., and Roux, K. H. 2009. Effects of long-term storage on soybean (Glycine max L.) proteins. J. Agric. Food Chem. 57:1312-1318.
• 13. Sharma, G. M., Roux, K. H., and Sathe, S. K. 2009. A sensitive and robust competitive enzyme-linked immunosorbent assay for Brazil nut (Bertholletia excelsa L.) detection. J. Agric. Food Chem. 57:769-776.
• 14. Robotham, J. M., Hoffman, G. G., Teuber, S. S., Beyer, K., Sampson, H. A., Sathe, S. K., and Roux, K. H. 2009. Linear IgE-epitope mapping and comparative structural homology modeling of hazelnut and English walnut 11S globulins. Mol. Immunology 46:2975-2984.
• 15. Sathe, S. K., Venkatachalam, M., Sharma, G. M., Kshirsagar, H. H., Teuber, S. S., and Roux, K. H. 2009. Solubilization and Electrophoretic characterization of select edible nut seed proteins. J. Agric. Food Chem. 57:7848-7856.
• 16. Tiwari, R. S., Venkatachalam, M., Sharma, G. M., Su, M., Roux, K. H., and Sathe, S. K. 2010. Effect of food matrix on amandin, almond (Prunus dulcis L.) major protein, immunorecognition and recovery. LWT- Food Science and Technology 43:675-683.
• 25. Sathe, S. K., Sharma, G. M., and Roux, K. H. 2008. Tree nut allergens In Tree Nuts: Composition, Phytochemicals, and Health Effects, Chapter 4, Alasalavar, C. and Shahidi, F. (eds.), Taylor and Francis, Boca Raton, FL, pp. 65-83. 26. Sathe, S. K. 2010. Protein solubility and functionality. (Invited contribution). A book chapter in a Food Protein Chemistry book edited by Prof. N. Hettiarachchy.
• 17. Robotham, J.M., Xia, L., Willison, L.N., Teuber, S.S., Sathe, S.K., Roux, K.H., 2010. Characterization of a cashew allergen, 11S globulin (Ana o 2), conformational epitope. Mol. Immunol. 47:1830-1838.
• 18. Xia, L., Willison, L.N., Porter, L., Robotham, J. M., Teuber, S. S., Sathe, S. K., and Roux, K. H. 2010. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association. Mol. Immunol. 47:1808-1816.
• 19. Sharma, G. M., Mundoma, C., Seavy, M., Roux, K. H., and Sathe, S. K. 2010. Purification and biochemical characterization of Brazil nut (Bertholletia excelsa L.) seed storage proteins. J. Agric. Food Chem. 58:5714-5723.
• 20. Sharma, G. M., Su, M., Joshi, A. U., Roux, K. H., and Sathe, S. K. 2010. Functional properties of select edible oilseed proteins. J. Agric. Food Chem. 58:5457-5464.
• 21. Kshirsagar, H. H., Fajer, P., Sharma, G. M., Roux, K. H., and Sathe, S. K. 2010. Biochemical and spectroscopic characterization of almond and cashew nut seed 11S legumins, amandin and anacardein. J. Agric. Food Chem. Accepted for publication.
• 22. Sharma, G. M., Irsigler, A., Rani, D., Ayuso, R., Ludmilla, B., Hugh, H. A., Roux, K. H., and Sathe, S. K. 2010. Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan [Carya illinoinensis (Wangenh.) K. Koch]. Submitted to J. Agric. Food Chem. Submitted.
• Non-refereed (All are invited contributions) 23. Maleki, S. J. and Sathe, S. K. 2006. The Effects of Processing Methods on Allergenic Properties of Food Proteins, In Food Allergy, Maleki, S. J., Burks, A. W., and Helm, R. M. (eds.), American Society for Microbiology, 1752 N Street N. W., Washington, DC 20036. pp 309-322.
• 24. Sathe, S. K., Monaghan, E. K., Kshirsagar, H. H., Venkatachalam, M. 2008. Chemical composition of edible nut seeds and its implications in human health. In Tree Nuts: Composition, Phytochemicals, and Health Effects, Chapter 2, Alasalavar, C. and Shahidi, F. (eds.), CRC Press, Taylor and Francis Group, Boca Raton, FL, pp. 11-35.