Progress 08/01/04 to 07/31/07
Outputs
Crustacean shellfish (shrimp) are commonly allergenic. Potentially hazardous shrimp residues may occur in other foods. A sandwich-type enzyme-linked immunosorbent assay (ELISA) for the detection of shrimp residues was developed using sheep and rabbit antisera with a detection limit of 1-2 ppm. The assay was highly specific except for expected weak levels of cross-reactivity with other crustacean species such as lobster and crayfish. An ELISA for the detection of mustard residues in processed foods was developed using polyclonal antisera raised against a mixture of three mustard seeds (yellow, brown, and oriental) in rabbits, goat, and sheep. The polyclonal antisera from rabbits and sheep were used in developing a specific and sensitive mustard ELISA capable of detecting mustard at 1 ppm. Some cross-reactivity was observed with cumin and rapeseed. The performance of the mustard ELISA was validated in salad dressing and frankfurter. Low recovery of mustard from dressing
was likely due to the precipitation of mustard proteins under acidic conditions. The recovery of mustard from frankfurter was >95%. A retail survey revealed the inability of the mustard ELISA to detect mustard in foods that have been subjected to extensive heat treatment. In addition, a publicly available database of the sequences of known allergens was placed on the web as Allergenonline version 7.0. Allergenonline version 7.0 is the first completely curated and peer-reviewed database available to scientists and governmental officials. Initially, all potential allergen sequences in the NCBI Entrez Protein database were screened. The peer review process involved assessing the quantity and quality of scientific information relating to the allergenicity of candidate allergens. Criteria were developed to evaluate the evidence based on in vivo and in vitro tests using subjects with clinically defined, relevant allergic disease. Published data were reviewed by a panel of 7 worldwide
scientific experts. Allergenonline Version 7.0 was posted on the web in January 2007 and contains 1250 protein sequences supported by published information demonstrating their allergenicity. Many isoforms are listed from some proteins while others are represented by a single sequence.
Impacts
The project has applicability for the food industry in protecting food-allergic consumers from undeclared residues of shrimp and mustard by developing and providing highly sensitive and specific methods to test food and to assess the effectiveness of allergen control programs including equipment cleaning measures. In addition, regulatory agencies will be able to use these methods to investigate complaints from shrimp- or mustard-allergic consumers who report reactions. The Allergenonline database is the first publicly available and fully peer-reviewed database of the amino acid sequences of protein allergens. The Allergenonline database can be used by researchers or regulators to evaluate the potentially allergenicity of proteins introduced into novel foods including genetically engineered foods. This peer-reviewed database increases the probability of identifying potentially cross-reactive proteins, thereby increasing the predictability of the safety assessments.
Publications
- Goodman, R. E. 2005. Assessing genetically modified crops to minimize the risk of increased food allergy: a review. Int. Arch. Allergy Immunol. 137:153-166.
- Taylor, S. L., Goodman, R. E., and Hefle, S. L. 2006. The development of safety assessment for genetically modified foods. Asia Pacific Biotech News 10:614-616.
- Taylor, S. L. 2006. Review of the development of methodology for evaluating the human allergenic potential of novel proteins. Molec. Nutr. Food Res. 50:604-609.
- Koppelman, S. J. and Hefle, S. L. (eds.). 2006. Detecting Allergens in Foods, Woodhead Publishing Ltd., Cambridge, England, 422 pp.
- Koppelman, S. J. and Hefle, S. L. 2006. Detecting soy, fish, and crustaceans in foods. In: Detecting Allergens in Foods, Koppelman, S. J. and Hefle, S. L. (eds.), Woodhead Publishing Ltd., Cambridge, England, pp. 273-290.
- Goodman, R., Ebisawa, M., Hefle, S., Sampson, H., van Ree, R., Vieths, S., Wise, J., and Taylor, S. L. 2007. Allergen Online, an improved, curated protein database for assessing the potential allergenicity of genetically modified organisms and novel food ingredients. Allergy 62 (Suppl. 83):106.
- Hefle, S. L., Taylor, S. L., and Bush, R. K. 2007. Seafood allergies. In: Wilderness Medicine, 5th edition, Auerbach, P. C. (ed.), Mosby/Elsevier, Philadelphia, PA, pp. 1559-1566.
- Lee, P. W. 2007. Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of mustard seed residues in processed foods. M. S. Thesis, University of Nebraska, (scheduled for completion in August, 2007).
- Estrada, C. 2005. Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of shrimp residues in processed foods. M.S. Thesis, University of Nebraska, 150 pp.
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Progress 10/01/05 to 09/30/06
Outputs
A mixture of different mustards and mustard flours was used to immunize rabbits, goats and sheep and antibodies were purified from their serum. The best immune responses were obtained in rabbits and goats. An enzyme-linked immunosorbent assay (ELISA) was developed using rabbit antibodies as the capture reagent and goat antibodies as detector reagent. Binding was visualized by addition of goat anti-rabbit IgG labeled with alkaline phosphatase and subsequent substrate addition. Validation of assay performance included testing sausage prepared with different known amounts of mustard flour (0-100 ppm), and cross reactivity assessment. The mustard ELISA has a limit of quantification of <1 ppm (1 ug/g) in sausage. This ELISA is very specific with observable cross-reactivity limited to rapeseed, a very closely related food. A peer-reviewed version of the AllergenOnline database was developed and will soon be posted as a publicly accessible resource at www.allergenonline.com.
Several very large protein databases were screened for potential allergens using several different strategies. The resulting database was curated by removing obvious irrelevant proteins identified by those approaches. The remaining 1557 protein sequences were reviewed by an international panel of scientific experts to arrive at a list of protein sequences that are considered to be allergens or putative allergens based upon review of supporting scientific information. Proteins available through May, 2005 were carried through this process. Currently, the large protein databases have been screened for new "allergen" entries entered since May 2005. The expert panel is reviewing those proteins to determine if sufficient evidence exists to add any of them to the existing database. The final peer-reviewed database will be released in mid-December 2006. The AllergenOnline database will contain the amino acid sequences of these known allergens along with query strategies for assessing the
degree of sequence homology of novel proteins to the allergens in the database.
Impacts
Errors can occur in food processing and preparation that can result in contamination of foods with residues of allergenic foods. Mustard is increasingly recognized as an allergenic food and is already on the list of commonly allergenic foods in Europe. The development of a sensitive and specific ELISA method for the detection of mustard residues gives food manufacturers a tool for assuring safety of their products, when using mustard in food applications. The AllergenOnline database of the amino acid sequences of known allergens is publicly available and has been used by researchers worldwide who are evaluating the potential allergenicity of proteins introduced into genetically modified foods and novel proteins being considered for use as food ingredients. The new database will be the first peer-reviewed and publicly available database of amino acid sequences of known allergens.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
The development of high quality antibodies for the mustard has begun. Rabbit, goat, and sheep antibodies are being produced and have been initially evaluated in an inhibition-type ELISA. These antibodies are a key reagent to developing a robust and east to use method for detection of mustard residues in other foods. The assay in development is a sandwich-type ELISA with a projected detection limit of 1 ppm mustard. This method will assist the food industry in assessing and implementing cleaning strategies that are effective at removing potentially hazardous mustard residues. The AllergenOnline database update is in progress to include newly identified and tested allergenic proteins that have been identified in the literature. An SQL database system has been developed to accept, store and allow restricted, structured access. An updated dataset has been compiled as the draft of version 6.0. This draft has been curated to remove illegitimate sequences (those clearly not
associated with allergy, sequences that were later identified as wrong) and include pertinent reference information. Security is built into the database with three levels of access, including one for the international allergy expert panel to review and enter recommendations for each entry. The curated version includes 1559 protein sequences (there were 1191 in version 5.0) that are being classified as allergens, putative allergens or non-allergens based a review of published studies evaluating IgE binding or clinical reactivity of the proteins using appropriately defined clinically allergic subjects. The review is being conducted with the assistance of an international panel of allergy experts. Scientific review is expected to be completed in August of 2006.
Impacts
This project has applicability for the food industry in protecting the food-allergic consumer from undeclared mustard allergenic residues in foods by providing a method to test their foods and equipment cleaning effectiveness. In addition, regulatory agencies can use the method to investigate complaints from mustard allergic consumers who report reactions. The AllergenOnline database is publicly available and has been used by researchers who are evaluating the potential allergenicity of proteins introduced into genetically modified crops, and proteins being considered for various uses in foods (personal communications). The updated database increases the probability of identifying potentially cross-reactive proteins, thereby increasing the predictability of the safety assessments.
Publications
- No publications reported this period
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Progress 08/01/04 to 07/31/05
Outputs
The development of immunoassays for the detection of shrimp allergenic residues in foods has been completed. These assays have been used extensively by our laboratory and the food industry to date to investigate shrimp residue contamination of foods, from the marketplace, and from consumer complaints and also industrial samples (oil use to fry shrimp and then other foods). The assays have detection limits of 1 - 2 ppm in a variety of foodstuffs. The assays are highly reproducible and specific, and have been used in battered products, sauces, and oils. These methods and the resulting protocols will assist the food industry in assessing and implementing cleaning strategies that are effective at removing potentially hazardous shrimp residues. The shrimp ELISA work has resulted in one masters thesis to date, with one journal article anticipated to be published also, in 2005.
Impacts
This project has applicability for the food industry in protecting the food-allergic consumer from undeclared soy and pecan allergenic residues in foods by providing methods to test their foods and equipment cleaning effectiveness. In addition, regulatory agencies can use the methods to investigate complaints from shrimp-allergic consumers who report reactions.
Publications
- Hefle, S.L., Taylor, S.L., and Bush, R.K. 2005. Seafood allergies. In: Wilderness Medicine, 5th Edition, Auerbach, P.C. (ed.), Macmillan, New York.
- Estrada, C. 2005. Development of an enzyme-linked immunosorbent assay ELISA) for the detection of shrimp residues in processed foods. Thesis (M.S.), University of Nebraska-Lincoln,150 pp.
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Progress 08/01/04 to 11/01/04
Outputs
The development of immunoassays for the detection of processed soy and whet allergenic residues in foods has begun. Antibodies against nine types of processed soy ingredients have just started being collected. These antibodies will be useful in improving our current soy ELISA in order to detect the more commonly used forms of soy ingredient in the food industry. The current soy assay has been used extensively by our laboratory and the food industry to date to investigate soy allergenic residue contamination of foods, both from the marketplace and as part of consumer complaints. High-quality whey antibodies have already been produced. The assays in development are sandwich-type ELISAs with projected detection limits of 1 ppm soy flour or minimally-processed soy ingredient, or 1 ppm whey, respectively. These methods will assist the food industry in assessing and implementing cleaning strategies that are effective at removing potentially hazardous soy residues. Work has
begun on the detection and identification of other allergens in legumes using proteomic analysis. Corn extrusion studies on fumonisin B1 have begun.
Impacts
This project has applicability for the food industry in protecting the food-allergic consumer from undeclared soy allergenic residues in foods by providing methods to test their foods and equipment cleaning effectiveness. In addition, regulatory agencies can use the methods to investigate complaints from soy and pecan allergic consumers who report reactions. The identification and isolation of allergenic food proteins contributes scientific information critical in trying to answer ascertain why people develop life-threatening allergic reactions to foods and other things, and to the safety of GMO crops. The evaluation of the corn extrusion process in reducing or eliminating the mycotoxins fumonisin B1 will be useful in developing food processing approaches in reducing the amount of this naturally occurring toxin in corn, and will result in great consumer safety due to reduced exposure to this toxin.
Publications
- No publications reported this period
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