- Email: [email protected]
Validated ELISA for detection of undeclared walnut residues in food
$248
Abstracts
720 from Identification and Cloning of Jug r 4, a Major Food Allergen EnglishWalnut Belongingto the Legumin Group S. S. Teuber I, W. R. Peterson ], S. Uratsu I, A. Dandekar ], K. H. Roux 2, S. K. Sathe2; 1University of California, Davis, CA, 2Florida State University, Tallahassee, FL. Food allergens in English walnut (Juglans regia) kernels are still incompletely characterized. We have found that sera from a majority of patients with a history of life-threatening systemic allergic reactions to walnut show IgE binding to legumin group seed storage proteins. We sought to clone one of the genes encoding a legumin group protein, express the fusion protein, and screen with sera from food-allergic patients with a history of severe, life-threatening reactions to walnut. Degenerate probes were used to obtain the cDNA by 5"- and 3"-RACE-PCR from walnut mRNA. The cDNA was subcloned into the pMAL vector and the recombinant fusion protein expressed in E. coll. The 1.5 kB insert encodes a polypeptide precursor protein with a predicted MW of approximately 56 kD. Excellent expression of the fusion protein was only obtained after deleting 96 bases of the 5" end, which corresponds to removing 32 amino acids from the hydrophobic N-terminus, and subcloning and expressing this fragment. 15 of 23 (65%) sera from patients with life-threatening reactions to walnut showed igE binding by immunoblot to the fusion protein. In conclusion, a gene encoding a walnut legumin precursor has been identified and validated as a major food allergen via analysis of the fusion protein and has tentatively been named Jug r 4.
Funding: University of California
721 Validatedin Food ELISAfor Detection of Undeclared Walnut Residues
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
versity of Arkansas for Medical Sciences, Little Rock, AR, 2Arkansas Children's Hospital, Little Rock, AR, 3Anatomy, University of Arkansas for Medical Sciences, Little Rock, AR, 4Mt. Sinai School of Medicine, New York, NY, 5Monsanto, St. Louis, MO, RATIONALE: 2D-proteomic mapping, immunoblotting and MS/MS analysis combined with sera of walnut allergic individuals were used to identify walnut proteins that bound IgE. METHODS: Utilizing amino acid sequence information from MS/MS analysis, specific degenerate primers were synthesized and PCR were used to amplify several gene fragments from a cDNA black walnut library. PCR products were ligated into the TA-vector and sequenced. The sequence of these fragments was used to design additional non-degenerated primers to obtain full length coding regions. RESULTS: A cDNA clone (749 bp), which codes a 234 amino acid protein revealed 83%, 82% and 81% protein sequence homology with rubber tree manganese superdoxide dismumtase (MnSOD), rice MnSOD and tobacco MnSOD, respectively. A second clone (1317 bp), which code a 338 amino acid protein, revealed 84% and 83% protein sequence homology with Pea and Tobacco glyceraldehydes (GAPDH), respectively. The coding sequences were put into pET 24a vector with His-tail and recombinant proteins expressed as single proteins. Using black walnut positive sera and SDS-PAGE/immunoblotting, 5/16 sera bound to SOD and 4/16 to GAPDH. Epitope mappings of these two allergens performed by the SPOTS techniques revealed four IgE-binding epitopes in SOD and four in GAPDH respectively. CONCLUSIONS: These results indicate that IgE from walnut allergic patients recognize walnut SOD and GAPDH in addition of Jug n I and Jug n2.
Funding: NIH L. Niemann, S. L. Here; Food Allergy Research and Resource Program, University of Nebraska, Lincoln, NE. RATIONALE: Tree nuts are responsible for some of the most severe, life-threatening allergic reactions, including several fatalities. In the US, hidden walnut or walnut residue is considered to be one of the most common reasons for allergic reactions to tree nuts. Rapid, sensitive methods for the detection of allergenic residues such as walnut are needed by the food industry to assess allergen control measures. METHODS: Walnuts (several varieties, raw and roasted) were washed, mixed, defatted, and ground to a powder. Rabbits and sheep were immunized and polyclonal antibodies purified from animal serum, Sheep antibodies were used as a capture reagent and rabbit antibodies were used as a detector reagent. Binding was visualized by addition of commercial goat anti-rabbit lgG labeled with alkaline phosphatase and subsequent substrate addition. Validation of assay performance included testing foods prepared with different known amounts of walnut on either pilot plant or industrial scale (0- 100 ppm) and cross reactivity assessment. RESULTS: The walnut ELISA has a detection limit of 1 ppm (1 ug/g) walnut; some cross reactivity was observed with hazelnut, pecan (close relative of walnut), and sesame seed, although more than 50 other foods and food ingredients did not register any cross-reactivity. CONCLUSIONS: This method gives food manufacturers a tool for assuring safety of their products for walnut-allergic individuals and will assist regulatory agencies in enforcement, thereby serving to better protect the walnutallergic consumer. This assay can also help improve labeling of food products and therefore positively affect quality of life for walnut-allergic consumers.
Funding: Food Allergy Research and Resource Program
722
Cloning,Identification,and Epitope-MappingTwo Black Walnut (Juglans Niger) Allergens
M. Ling 1,2, J. Ye 3, K. Beyer4, G. Cockrell 1.2, G. A. Bannon 5, J. S. Stanley 1,2, R. M. Helm L,2, H. A. Sampson4, A. W. Burksl,2; 1Pediatrics, Uni-
723 Recombinant Hazelnut LTP,Cur a 8: A Useful Tool in the in vitro Diagnosisof Hazelnut Allergy F. Schocker I, D. Liittkopf2, A. Cister6-Bahfma3, E. Enrique 3, J. H. Akkerdaas 4, R. van Ree 4, S. Vieths2, W. M. Beckerl; IBiochemical and Molecular Allergology, Research Center Borstel, Borstel, GERMANY, 2paulEhrlich-lnstitut, Langen, GERMANY, 3Institut Universitari Dexeus, Barcelona, SPAIN, 4CLB, Sanquin, Amsterdam, NETHERLANDS. RATIONALE: Lipid transfer proteins (LTP) are known as food allergens which can elicit severe allergenic reactions mainly in the Mediterranean area. Recently, we were able to clone the full length cDNA of hazelnut LTP and express its recombinant form (denominated as Cor a 8 according to the WHO/IUIS Allergen Nomenclature Subcommittee). The purpose of this study was to investigate the lgE-reactivity to the recombinant Cor a 8 in comparison to hazelnut extract. METHODS: Cor a 8 was expressed in E. colt and purified by His-tag chromatography. Sera from 26 Spanish patients with hazelnut allergy were tested by performing immunoblots with r Cor a 8 and hazelnut extract. Immunoblot inhibition was performed to investigate immunological properties of natural and recombinant Cot a 8. RESULTS: Eighteen out of 26 Spanish sera showed IgE-reactivity to n Cor a 8 in hazelnut extract of which 13 patients were monosensitized to Cor a 8. These results could be fully confirmed by using the recombinant allergen. Immunoblot inhibition demonstrated epitope similarity of natural and recombinant Cot a 8. CONCLUSIONS: LTP is a major allergen in Spanish patients with hazelnut allergy. As demonstrated by means of immunoblot and immunoblot inhibition recombinant hazelnut LTP has the same biologic activity as natural LTP. Since quality problems with allergen extracts from foods still exist and LTPs are known as potentially severe food allergens, the use of r Cor a 8 may improve the in vitro diagnosis of food allergy.
Funding: Self-funded
Abstracts
720 from Identification and Cloning of Jug r 4, a Major Food Allergen EnglishWalnut Belongingto the Legumin Group S. S. Teuber I, W. R. Peterson ], S. Uratsu I, A. Dandekar ], K. H. Roux 2, S. K. Sathe2; 1University of California, Davis, CA, 2Florida State University, Tallahassee, FL. Food allergens in English walnut (Juglans regia) kernels are still incompletely characterized. We have found that sera from a majority of patients with a history of life-threatening systemic allergic reactions to walnut show IgE binding to legumin group seed storage proteins. We sought to clone one of the genes encoding a legumin group protein, express the fusion protein, and screen with sera from food-allergic patients with a history of severe, life-threatening reactions to walnut. Degenerate probes were used to obtain the cDNA by 5"- and 3"-RACE-PCR from walnut mRNA. The cDNA was subcloned into the pMAL vector and the recombinant fusion protein expressed in E. coll. The 1.5 kB insert encodes a polypeptide precursor protein with a predicted MW of approximately 56 kD. Excellent expression of the fusion protein was only obtained after deleting 96 bases of the 5" end, which corresponds to removing 32 amino acids from the hydrophobic N-terminus, and subcloning and expressing this fragment. 15 of 23 (65%) sera from patients with life-threatening reactions to walnut showed igE binding by immunoblot to the fusion protein. In conclusion, a gene encoding a walnut legumin precursor has been identified and validated as a major food allergen via analysis of the fusion protein and has tentatively been named Jug r 4.
Funding: University of California
721 Validatedin Food ELISAfor Detection of Undeclared Walnut Residues
J ALLERGY CLIN IMMUNOL FEBRUARY 2003
versity of Arkansas for Medical Sciences, Little Rock, AR, 2Arkansas Children's Hospital, Little Rock, AR, 3Anatomy, University of Arkansas for Medical Sciences, Little Rock, AR, 4Mt. Sinai School of Medicine, New York, NY, 5Monsanto, St. Louis, MO, RATIONALE: 2D-proteomic mapping, immunoblotting and MS/MS analysis combined with sera of walnut allergic individuals were used to identify walnut proteins that bound IgE. METHODS: Utilizing amino acid sequence information from MS/MS analysis, specific degenerate primers were synthesized and PCR were used to amplify several gene fragments from a cDNA black walnut library. PCR products were ligated into the TA-vector and sequenced. The sequence of these fragments was used to design additional non-degenerated primers to obtain full length coding regions. RESULTS: A cDNA clone (749 bp), which codes a 234 amino acid protein revealed 83%, 82% and 81% protein sequence homology with rubber tree manganese superdoxide dismumtase (MnSOD), rice MnSOD and tobacco MnSOD, respectively. A second clone (1317 bp), which code a 338 amino acid protein, revealed 84% and 83% protein sequence homology with Pea and Tobacco glyceraldehydes (GAPDH), respectively. The coding sequences were put into pET 24a vector with His-tail and recombinant proteins expressed as single proteins. Using black walnut positive sera and SDS-PAGE/immunoblotting, 5/16 sera bound to SOD and 4/16 to GAPDH. Epitope mappings of these two allergens performed by the SPOTS techniques revealed four IgE-binding epitopes in SOD and four in GAPDH respectively. CONCLUSIONS: These results indicate that IgE from walnut allergic patients recognize walnut SOD and GAPDH in addition of Jug n I and Jug n2.
Funding: NIH L. Niemann, S. L. Here; Food Allergy Research and Resource Program, University of Nebraska, Lincoln, NE. RATIONALE: Tree nuts are responsible for some of the most severe, life-threatening allergic reactions, including several fatalities. In the US, hidden walnut or walnut residue is considered to be one of the most common reasons for allergic reactions to tree nuts. Rapid, sensitive methods for the detection of allergenic residues such as walnut are needed by the food industry to assess allergen control measures. METHODS: Walnuts (several varieties, raw and roasted) were washed, mixed, defatted, and ground to a powder. Rabbits and sheep were immunized and polyclonal antibodies purified from animal serum, Sheep antibodies were used as a capture reagent and rabbit antibodies were used as a detector reagent. Binding was visualized by addition of commercial goat anti-rabbit lgG labeled with alkaline phosphatase and subsequent substrate addition. Validation of assay performance included testing foods prepared with different known amounts of walnut on either pilot plant or industrial scale (0- 100 ppm) and cross reactivity assessment. RESULTS: The walnut ELISA has a detection limit of 1 ppm (1 ug/g) walnut; some cross reactivity was observed with hazelnut, pecan (close relative of walnut), and sesame seed, although more than 50 other foods and food ingredients did not register any cross-reactivity. CONCLUSIONS: This method gives food manufacturers a tool for assuring safety of their products for walnut-allergic individuals and will assist regulatory agencies in enforcement, thereby serving to better protect the walnutallergic consumer. This assay can also help improve labeling of food products and therefore positively affect quality of life for walnut-allergic consumers.
Funding: Food Allergy Research and Resource Program
722
Cloning,Identification,and Epitope-MappingTwo Black Walnut (Juglans Niger) Allergens
M. Ling 1,2, J. Ye 3, K. Beyer4, G. Cockrell 1.2, G. A. Bannon 5, J. S. Stanley 1,2, R. M. Helm L,2, H. A. Sampson4, A. W. Burksl,2; 1Pediatrics, Uni-
723 Recombinant Hazelnut LTP,Cur a 8: A Useful Tool in the in vitro Diagnosisof Hazelnut Allergy F. Schocker I, D. Liittkopf2, A. Cister6-Bahfma3, E. Enrique 3, J. H. Akkerdaas 4, R. van Ree 4, S. Vieths2, W. M. Beckerl; IBiochemical and Molecular Allergology, Research Center Borstel, Borstel, GERMANY, 2paulEhrlich-lnstitut, Langen, GERMANY, 3Institut Universitari Dexeus, Barcelona, SPAIN, 4CLB, Sanquin, Amsterdam, NETHERLANDS. RATIONALE: Lipid transfer proteins (LTP) are known as food allergens which can elicit severe allergenic reactions mainly in the Mediterranean area. Recently, we were able to clone the full length cDNA of hazelnut LTP and express its recombinant form (denominated as Cor a 8 according to the WHO/IUIS Allergen Nomenclature Subcommittee). The purpose of this study was to investigate the lgE-reactivity to the recombinant Cor a 8 in comparison to hazelnut extract. METHODS: Cor a 8 was expressed in E. colt and purified by His-tag chromatography. Sera from 26 Spanish patients with hazelnut allergy were tested by performing immunoblots with r Cor a 8 and hazelnut extract. Immunoblot inhibition was performed to investigate immunological properties of natural and recombinant Cot a 8. RESULTS: Eighteen out of 26 Spanish sera showed IgE-reactivity to n Cor a 8 in hazelnut extract of which 13 patients were monosensitized to Cor a 8. These results could be fully confirmed by using the recombinant allergen. Immunoblot inhibition demonstrated epitope similarity of natural and recombinant Cot a 8. CONCLUSIONS: LTP is a major allergen in Spanish patients with hazelnut allergy. As demonstrated by means of immunoblot and immunoblot inhibition recombinant hazelnut LTP has the same biologic activity as natural LTP. Since quality problems with allergen extracts from foods still exist and LTPs are known as potentially severe food allergens, the use of r Cor a 8 may improve the in vitro diagnosis of food allergy.
Funding: Self-funded