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Detection Of Peanut Allergens In Breast Milk and Saliva
AB114 Abstracts
398
SUNDAY
Utility Of Probability Curves Using 3gAllergy For Diagnosis Of Wheat Allergy Sakura Sato1, Kiyotake Ogura1, Yasunori Sato2, Motohiro Ebisawa, MD, PhD, FAAAAI1; 1Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japan, 2Department of Biostatistics, Clinical Research Center Chiba University. RATIONALE: Previously we presented the utility of allergen specific IgE (sIgE) measurements by 3gAllergy using probability curves for diagnosis of hen’s egg and cow’s milk allergy. In this study, we assessed the utility of probability curves in the diagnosis of wheat allergy. METHODS: Serum samples were collected from 330 children (mean age 10.5 months) suspected of having wheat allergy. Allergen sIgE testing for wheat (W) and Gluten (G) was performed using the IMMULITEÒ 2000 3gAllergyTM and ImmunoCAPÒ sIgE assays. Final diagnosis of food allergy (FA) was confirmed by oral food challenge (OFC) and convincing allergic reactions to wheat products; acquisition of tolerance was defined as patients who had ingested 100 g of Japanese wheat noodle without any symptoms. Serum samples were drawn 6 months before the FA diagnosis and then stored frozen. RESULTS: Of the 330 patients, 49 children were diagnosed with W allergy. W and G sIgE measurements by 3g were correlated with CAP W (W: r50.91, G: r50.93, p<0.0001); In comparison to CAP, 3g W values were likely to be lower than Cap W. Probability curves using the sIgE values from the FA positive negative groups were established using logistic regression analysis, and the 95% predictive decision point were determined by 3g (W: 234 IUA/mL, G: 92 IUA/mL.) The 95% decision point for W by CAP was not determined because sIgE results exceeded the range of the assay. CONCLUSIONS: Measurement of sIgE to W and G using 3g and the PC are beneficial for diagnosis of W allergy.
399
Skin Prick Test and Specific IgE To Purified Peanut Allergens Are Related To The Age Of Onset Of Sympstons Dr. Maria Salas, MD, PhD1, Dr. Francisca Gomez, MD, PhD2, Dr. Ana Aranda, PhD3, Dr. Carmen Rondon, MD, PhD1, Dr. Natalia BlancaL opez, MD, PhD4, Dr. Gabriela Canto, MD, PhD4, Dr. Maria J Torres, MD, PhD1, Dr. Cristobalina Mayorga, PhD3, Dr. Miguel Blanca, MD, PhD1, Maria Isabel Sanchez Rivas5; 1Allergy Service, Carlos Haya Hospital, Malaga, Spain, 2Allergy Service, Carlos Haya Hospital, Spain, 3Research Laboratory, Carlos Haya Hospital-FIMABIS, Malaga, Spain, 4Allergy Service, Infanta Leonor Hospital, Madrid, Spain, 5Allergy Service Carlos Haya Hospital, Malaga, Spain. RATIONALE: We aim to analyse changes in the skin prick test (SPT) to peanut and IgE levels to Ara h1; Ara h2; Arah3; Ara h9 and Pru p3 in relationship with the age of patients and the onsent of symptons. METHODS: We studied 167 peanut allergic patients confirmed by SPT or prick by prick to fresh extract, in vitro specific IgE and/or a double blind placebo control challenge with peanut. Patients were divided into 3 ages groups: 15-30, 31-45 and 46-80. IgE levels were determined by ELISA to Ara h1, Ara h2, Ara h3, Ara h9 and Pru p3. RESULTS: Thirty-three patients evaluated in the first group (54%) were positive to peanut in SPT, 34 (42,50%) in second group and 4 (15%) in the last group. ELISA showed 50% positivity to Ara h1, Ara h2; Ara h3, 60% to Pru p3 and 80% to Ara h9 in the first group. 40% to Ara h1, Ara h3, 50% to Ara h9 and Pru p3 and less than 30% to Arah2 in the second group. In the third group IgE levels fall below 20% to Ara h2, Ara h3 and Ara h9. Ara h1 and Pru p3 were not detected. CONCLUSIONS: We observed a decrease in the reactivity response of SPT to peanut and IgE levels in the third age group with and onset of symptoms over tan ten years. Levels to Ara h 2 were lower on median ages, although Ara h9 and Pru p3 were similar in the the first groups.
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
400
The Relationship Between Mitochondrial Haplogroups Variant on Children with Cow Milk Allergy Expressed As Atopic Dermatitis and Gastrointestinal Disease Dr. Juan Carlos Muino, MD, PhD, FAAAAI1, Dr. Raul Boudet1, Dr. Maria Chaig1, Dr. Roberto Chaig1, Prof. Nelida Gerez2, Prof. Juan Carlos Copioli1; 1FAC CS MED UNC, Cordoba, Argentina, 2FAC CS MED UNC, Cordoba, Argentina. RATIONALE: Genotypes associated to cow’s milk allergy are unknown. They have not been replicated in independent population, and could be responsible for the marked variability in individual clinical response to cow milk proteins.The objective was to characterize haplogroups of the D-Loop region of mitochondrial DNA in a group of children allergic to cow’s milk in order to arrive to a better knowledge of biological and genetic heritability in the etiology of the disease METHODS: .We studied 41 children of both sex aged 0 – 2 years, 11 allergic to cow’s milk demonstrated by challenge and 30 healthy subjects (controls), from the urban area of Rio Cuarto City, Cordoba, Argentina. We performed Analysis of variants of D-loop region of the mitochondrial genome. The D-Loop region HVI, II and II of the mitochondrial genome was amplified by PCR, for which we used specific primers. Phylogenetic analysis was calculated using the program CLUSTAL OMEGA, the Neighbor–Joining, BLOSUM62 with data studied and recorded by JukesCantor and then with Kimura-2 RESULTS: The cow milk allergic patients were divided in: (a) Atopic Dermatitis (AD) + Gastrointestinal Disease (GID) (n: 6) and (b) Rhinitis and Asthma (n: 5). We found the non described variant in transition of haplogroups, T16519C associated with (a) in 6/6 cases when compared with (b) negative in 5/5 cases and the control group (6/30), p5 0, 0312, RR: 2,900 CONCLUSIONS: These features suggest that this variant probably increases the possibility of suffering cow milk allergy associated with AD +GID.
401
Detection Of Peanut Allergens In Breast Milk and Saliva Dr. Kelli M. Rose, MD1, Christian Plaisance2, Casey C. Grimm, PhD2, Hsiaopo Cheng, MS2, Tysheena Charles, MS2, Saeed A. Jortani3, Soheila J. Maleki, PhD2; 1Tulane University, New Orleans, LA, 2USDA-ARS-SRRC, New Orleans, LA, 3University of Louisville, Louisville, KY. RATIONALE: Infants have been reported to react to peanut upon their first known exposure and peanut proteins have been detected in breast milk. Identification of the allergens or fragments thereof in breast milk may allow us to determine if they are sensitizing or tolerizing. METHODS: Various immunoassays were optimized and utilized to analyze for the presence of peanut proteins in breast milk. Breast milk samples from healthy lactating mothers were collected following a 48 hour peanut fast and spiked with known amounts of peanuts and subjected to immunoprecipitation, SDS-PAGE, western blot and ELISA with antipeanut, anti-Ara h 1, 2 and 3 antibodies. Mass spectrometry was performed to identify peanut peptides in breast milk. RESULTS: We found that we were able to detect peanut allergen, Ara h 1, Ara h 2 and Ara h3, in peanut-spiked breast milk and saliva at nM levels. We were able to identify specific peanut peptides of Ara h 1, Ara h 2 and Ara h 3/4 using mass spectrometry. CONCLUSIONS: The fact that allergic proteins or peptides survive in digestive enzymes, and are likely to be secreted in biological fluids indicates they are most likely the sensitizing or tolerizing agents within an allergic food. Developing methods to detect these allergens in breast milk is a preliminary step in identification of allergens or fragments thereof that are secreted and may contribute to the original development of allergic disease.
398
SUNDAY
Utility Of Probability Curves Using 3gAllergy For Diagnosis Of Wheat Allergy Sakura Sato1, Kiyotake Ogura1, Yasunori Sato2, Motohiro Ebisawa, MD, PhD, FAAAAI1; 1Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japan, 2Department of Biostatistics, Clinical Research Center Chiba University. RATIONALE: Previously we presented the utility of allergen specific IgE (sIgE) measurements by 3gAllergy using probability curves for diagnosis of hen’s egg and cow’s milk allergy. In this study, we assessed the utility of probability curves in the diagnosis of wheat allergy. METHODS: Serum samples were collected from 330 children (mean age 10.5 months) suspected of having wheat allergy. Allergen sIgE testing for wheat (W) and Gluten (G) was performed using the IMMULITEÒ 2000 3gAllergyTM and ImmunoCAPÒ sIgE assays. Final diagnosis of food allergy (FA) was confirmed by oral food challenge (OFC) and convincing allergic reactions to wheat products; acquisition of tolerance was defined as patients who had ingested 100 g of Japanese wheat noodle without any symptoms. Serum samples were drawn 6 months before the FA diagnosis and then stored frozen. RESULTS: Of the 330 patients, 49 children were diagnosed with W allergy. W and G sIgE measurements by 3g were correlated with CAP W (W: r50.91, G: r50.93, p<0.0001); In comparison to CAP, 3g W values were likely to be lower than Cap W. Probability curves using the sIgE values from the FA positive negative groups were established using logistic regression analysis, and the 95% predictive decision point were determined by 3g (W: 234 IUA/mL, G: 92 IUA/mL.) The 95% decision point for W by CAP was not determined because sIgE results exceeded the range of the assay. CONCLUSIONS: Measurement of sIgE to W and G using 3g and the PC are beneficial for diagnosis of W allergy.
399
Skin Prick Test and Specific IgE To Purified Peanut Allergens Are Related To The Age Of Onset Of Sympstons Dr. Maria Salas, MD, PhD1, Dr. Francisca Gomez, MD, PhD2, Dr. Ana Aranda, PhD3, Dr. Carmen Rondon, MD, PhD1, Dr. Natalia BlancaL opez, MD, PhD4, Dr. Gabriela Canto, MD, PhD4, Dr. Maria J Torres, MD, PhD1, Dr. Cristobalina Mayorga, PhD3, Dr. Miguel Blanca, MD, PhD1, Maria Isabel Sanchez Rivas5; 1Allergy Service, Carlos Haya Hospital, Malaga, Spain, 2Allergy Service, Carlos Haya Hospital, Spain, 3Research Laboratory, Carlos Haya Hospital-FIMABIS, Malaga, Spain, 4Allergy Service, Infanta Leonor Hospital, Madrid, Spain, 5Allergy Service Carlos Haya Hospital, Malaga, Spain. RATIONALE: We aim to analyse changes in the skin prick test (SPT) to peanut and IgE levels to Ara h1; Ara h2; Arah3; Ara h9 and Pru p3 in relationship with the age of patients and the onsent of symptons. METHODS: We studied 167 peanut allergic patients confirmed by SPT or prick by prick to fresh extract, in vitro specific IgE and/or a double blind placebo control challenge with peanut. Patients were divided into 3 ages groups: 15-30, 31-45 and 46-80. IgE levels were determined by ELISA to Ara h1, Ara h2, Ara h3, Ara h9 and Pru p3. RESULTS: Thirty-three patients evaluated in the first group (54%) were positive to peanut in SPT, 34 (42,50%) in second group and 4 (15%) in the last group. ELISA showed 50% positivity to Ara h1, Ara h2; Ara h3, 60% to Pru p3 and 80% to Ara h9 in the first group. 40% to Ara h1, Ara h3, 50% to Ara h9 and Pru p3 and less than 30% to Arah2 in the second group. In the third group IgE levels fall below 20% to Ara h2, Ara h3 and Ara h9. Ara h1 and Pru p3 were not detected. CONCLUSIONS: We observed a decrease in the reactivity response of SPT to peanut and IgE levels in the third age group with and onset of symptoms over tan ten years. Levels to Ara h 2 were lower on median ages, although Ara h9 and Pru p3 were similar in the the first groups.
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
400
The Relationship Between Mitochondrial Haplogroups Variant on Children with Cow Milk Allergy Expressed As Atopic Dermatitis and Gastrointestinal Disease Dr. Juan Carlos Muino, MD, PhD, FAAAAI1, Dr. Raul Boudet1, Dr. Maria Chaig1, Dr. Roberto Chaig1, Prof. Nelida Gerez2, Prof. Juan Carlos Copioli1; 1FAC CS MED UNC, Cordoba, Argentina, 2FAC CS MED UNC, Cordoba, Argentina. RATIONALE: Genotypes associated to cow’s milk allergy are unknown. They have not been replicated in independent population, and could be responsible for the marked variability in individual clinical response to cow milk proteins.The objective was to characterize haplogroups of the D-Loop region of mitochondrial DNA in a group of children allergic to cow’s milk in order to arrive to a better knowledge of biological and genetic heritability in the etiology of the disease METHODS: .We studied 41 children of both sex aged 0 – 2 years, 11 allergic to cow’s milk demonstrated by challenge and 30 healthy subjects (controls), from the urban area of Rio Cuarto City, Cordoba, Argentina. We performed Analysis of variants of D-loop region of the mitochondrial genome. The D-Loop region HVI, II and II of the mitochondrial genome was amplified by PCR, for which we used specific primers. Phylogenetic analysis was calculated using the program CLUSTAL OMEGA, the Neighbor–Joining, BLOSUM62 with data studied and recorded by JukesCantor and then with Kimura-2 RESULTS: The cow milk allergic patients were divided in: (a) Atopic Dermatitis (AD) + Gastrointestinal Disease (GID) (n: 6) and (b) Rhinitis and Asthma (n: 5). We found the non described variant in transition of haplogroups, T16519C associated with (a) in 6/6 cases when compared with (b) negative in 5/5 cases and the control group (6/30), p5 0, 0312, RR: 2,900 CONCLUSIONS: These features suggest that this variant probably increases the possibility of suffering cow milk allergy associated with AD +GID.
401
Detection Of Peanut Allergens In Breast Milk and Saliva Dr. Kelli M. Rose, MD1, Christian Plaisance2, Casey C. Grimm, PhD2, Hsiaopo Cheng, MS2, Tysheena Charles, MS2, Saeed A. Jortani3, Soheila J. Maleki, PhD2; 1Tulane University, New Orleans, LA, 2USDA-ARS-SRRC, New Orleans, LA, 3University of Louisville, Louisville, KY. RATIONALE: Infants have been reported to react to peanut upon their first known exposure and peanut proteins have been detected in breast milk. Identification of the allergens or fragments thereof in breast milk may allow us to determine if they are sensitizing or tolerizing. METHODS: Various immunoassays were optimized and utilized to analyze for the presence of peanut proteins in breast milk. Breast milk samples from healthy lactating mothers were collected following a 48 hour peanut fast and spiked with known amounts of peanuts and subjected to immunoprecipitation, SDS-PAGE, western blot and ELISA with antipeanut, anti-Ara h 1, 2 and 3 antibodies. Mass spectrometry was performed to identify peanut peptides in breast milk. RESULTS: We found that we were able to detect peanut allergen, Ara h 1, Ara h 2 and Ara h3, in peanut-spiked breast milk and saliva at nM levels. We were able to identify specific peanut peptides of Ara h 1, Ara h 2 and Ara h 3/4 using mass spectrometry. CONCLUSIONS: The fact that allergic proteins or peptides survive in digestive enzymes, and are likely to be secreted in biological fluids indicates they are most likely the sensitizing or tolerizing agents within an allergic food. Developing methods to detect these allergens in breast milk is a preliminary step in identification of allergens or fragments thereof that are secreted and may contribute to the original development of allergic disease.