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67 A Method of Reducing Allergenicity of Legume ProteinsNaveen Arora 1 , Ramkrashan Kasera 2 , Anand Singh, PhD 3 ,Shakuntala Lavasa

4, Komerla Nagendra

5;

1CSIR- IGIB, Delhi, India,

2CSIR-Institute of Genomics and Integrative Biology, India,

3Institute of

Genomics and Integrative Biology, DELHI, India, 4

USLavasa Medical& Research Center, India,

5Bengaluru Allergy Centre, India.

RATIONALE: Legumes are implicated in IgE mediated food allergy in

different countries. The present study aimed to investigate the effect of enzymatic hydrolysison allergenicityofkidneybean,black gram andpeanut.METHODS: The extracts were subjected to sequential enzymatic treat-ment by Alcalase and avorzyme. The allergenicity of hydrolysates weredetermined by ELISA and SPT. Allergenic potential of legume proteinswere assessed by ELISA inhibition and basophil histamine release.RESULTS: Hydrolysis reduced thenumber of IgEbinding fractionsof thethree legumes- kidney bean, black gram and peanut. Specic IgE bindingwas reduced 62%, 87% and 92% in the hyrolysates of kidney bean, black gram and peanut, respectively after 8 h of hydrolysis (p < 0.01). Alcalasetreatment alone increased the IC50 value of kidney bean, black gram andpeanut to 4, 8.5 and 123 folds respectively. Sequential hydrolysis usingboth alcalase andavourzymeincreased theIC50 of thekidney bean, black gram and peanut to 25, 368 and 393 folds respectively further as comparedto single enzyme treatment. Basophil histamine release showed reducedallergenicity of the hydrolyzed proteins. Signicant reduction in thebiopotency of hydrolyzed protein of kidney bean, black gram and peanutwas observed in SPT where 6/13 kidney bean sensitive individuals, 8/13blackgram sensitive individuals and 9/15peanut sensitive individuals werefound SPT negative to their respective hydrolysates.CONCLUSIONS: Sequential hydrolysis by alcalase and avourzymereduced the allergenicity of legume proteins. The method has potential toreduce allergenicity of food extracts.

68 Comparative Analysis of IgE Binding Proteins in GM and Non-GM Rice Varieties Using Atopic Patients SeraChandni Mathur 1 , P. C Kathuria

2, Shakuntala Lavasa

3, Pushpa Dahiya

4,

Anand Singh, PhD5;

1Institute of Genomics & Integrative Biology, Delhi,

India, 2

National Allergy Centre, India, 3

USLavasa Medical & ResearchCenter, India,

4M.D University, India,

5Institute of Genomics and Integra-

tive Biology, Delhi, India.RATIONALE: Food allergy is recognized as IgE-mediated immuneresponse. Safety issues regarding foods derived from genetically modied(GM) plants are central to their acceptance into the food supply and itscommercial production.METHODS: Three GM rice varieties containing cry protein Cry 1ab, Cry1ac and Cry 1c along with their nativevariety were selected for the presentinvestigation. All the seed powders were processed for protein extractionand estimation. Extracts were subjected to SDS-PAGE for protein prol-ing. Simulated Gastric Fluid Digestion (SGF) was also done for all seedpowders. IgE binding protein bands in rice were observed throughImmunoblot with sera of allergic patients. Bioinformatic analysis of abovetransgenic proteins across databases was also performed.RESULTS: Protein content in non-GM rice was 0.9 mg/ml while in GM

rice varieties it varied from 0.95 1.2 mg/ml. Proteins fractionated in 11-14bands in allvarieties within a range of9-81 kD.Native rice andGM ricewith Cry 1Ab subjected to SGF showed a very faint appearance of smeararound9kD. Rice variety withCry 1Abhad faint proteins around27 and47kD. IgE binding protein bands observed in rice varieties were aggregatingaround 37-47 kD. Comparative analysis across different databases yieldedthat cry proteins used in GM Rice showed sequence identity of < 27 % .CONCLUSIONS: It is suspected that no major detectable difference wasobserved in GM and non-GM rice varieties.

69 Analysis of the Association Between Food Allergy andSensitization to Ltp and ProfilinFelicia Berroa 1 , M Jose Goikoetxea, PhD, MD

2, Marta M.

Ferrer, MD, PhD, FAAAAI2, Paula Cabrera-Freitag, MD, PhD

3, Gracia

Javaloyes, MD, PhD1, Maria L Sanz, MD, PhD

1, Gabriel Gastaminza,

MD, PhD1;

1Department of Allergy, Clinica Universidad de Navarra,

Spain, 2

Department of Allergy, Clinica Universidad de Navarra, Pam-

plona, Spain, 3

Clinica Universidad de Navarra, Pamplona, Spain.RATIONALE: LTP and prolin panallergens are associated with foodallergy (FA). The aim of our study was to evaluate the association of suffering oral allergy syndrome (OAS) and systemic symptoms (SS)according to sensitization to LTP or prolin.METHODS: Retrospective analysis of 238 patients in whom skin prick test (SPT) to palm prolin and peach LTP(ALK-Abell o, Madrid, Spain)was performed. Specic IgE rPhl p 12 and rPru p 3 were analyzed byImmunoCAP (Phadia, Sweden) from serum obtained on the day of consultation. The association to present OAS or SS after ingestion of plant-food was studied by Chi-square and Odds Ratio(OR) calculation.RESULTS: Out of 238 patients, 72 (30%) had FA: 36 presented OAS, 20patients SS and 16 OAS and SS. Eighty-three patients had positve SPT toprolin and/or LTP (42 prolin, 29 LTP and 12 both panallergens).Skinsensitization to LTP was associated with: OAS (OR: 3.279 [1.595-6.741];p 5 0.001) and SS (OR: 5.664[2.598-12.349]; p < 0.001). Neither LTP-SPTnor rPru p 3 were associated with OAS without SS. A positive result torPrup3 by ImmunoCAP was associated with OAS, only in those patientssuffering also from SS. ImmunoCAP (OR: 2,568 [1,254-5,258]; p 5 0.008)but not SPT (OR: 2.206[1.121-4.342]; p 5 0.020) to prolin were associ-ated with OAS without SS.CONCLUSIONS: Having positive SPTand/or ImmunoCAP is associatedwith having systemic symptoms with or without OAS. When OAS ispresent without systemic symptoms is associated only with ImmunoCAPto rPhl p 12.

70 Structure and Function of the Peanut Panallergen Ara h 8Barry K. Hurlburt, PhD 1 , Lesa Celeste, PhD 2 , Karolina A.Majorek

3, Jane McBride

4, Soheila J. Maleki, PhD

1, Wladek

Minor, PhD3, Maksymilian Chruszcz, PhD

5;

1USDA-ARS-SRRC, New

Orleans, LA, 2 university of south carolina, columbia, SC, 3 University of Virginia, Charlottesville, VA,

4usda-ars-srrc, new orleans, LA,

5University

of South Carolina, Columbia, SC.RATIONALE: Ara h 8 is hypothesized to be the panallergen responsiblefor oral allergy syndrome between birch pollen and peanut. In addition, itmay also be a signaling molecule carrier. We sought to investigate thesenotions with structural, and biochemical characterization.METHODS: Recombinant Ara h 8 was expressed in E. coli and puried.The structurewas determinedusingX-ray crystallography.Ligand bindingwas tested by displacement of bounduorescent 8-anilo-1-naphthalenesul-fonic acid.RESULTS: Purication by a combination of heat treatment, ammoniumsulfateprecipitation and hydrophobic interaction chromatography yielded >95%pureArah 8. Thecrystal structure wassolvedby molecular replacement

andrened at1.7 A resolution. The overallfoldof Arah 8 wasvery similar toBet v 1, aswell asBet v 1 homologsApi g 1 (celery), Gly m 4 (soy),andPruav 1 (cherry). Physiologically-relevant ligands have been identied.CONCLUSIONS: Our results support both the idea that Ara h 8 could becontributing to oral allergy syndrome between birchpollen and peanut, andthe hypothesis that Bet v 1-like proteins could be transporters of signalingmolecules important for plant development.

J ALLERGY CLIN IMMUNOL

VOLUME 131, NUMBER 2Abstracts AB19

http://dx.doi.org/10.1016/j.jaci.2012.12.749http://dx.doi.org/10.1016/j.jaci.2012.12.749http://dx.doi.org/10.1016/j.jaci.2012.12.749http://dx.doi.org/10.1016/j.jaci.2012.12.749