J ALLERGY CLIN IMMUNOL

VOLUME 131, NUMBER 2

Abstracts AB19

SATURDAY

67 A Method of Reducing Allergenicity of Legume Proteins

Naveen Arora1, Ramkrashan Kasera2, Anand Singh, PhD3,

Shakuntala Lavasa4, Komerla Nagendra5; 1CSIR- IGIB, Delhi, India,2CSIR-Institute of Genomics and Integrative Biology, India, 3Institute of

Genomics and Integrative Biology, DELHI, India, 4USLavasa 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 hydrolysis on allergenicityof kidney bean, black gramandpeanut.

METHODS: The extracts were subjected to sequential enzymatic treat-

ment by Alcalase and flavorzyme. The allergenicity of hydrolysates were

determined by ELISA and SPT. Allergenic potential of legume proteins

were assessed by ELISA inhibition and basophil histamine release.

RESULTS: Hydrolysis reduced the number of IgE binding fractions of the

three legumes- kidney bean, black gram and peanut. Specific IgE binding

was reduced 62%, 87% and 92% in the hyrolysates of kidney bean, black

gram and peanut, respectively after 8 h of hydrolysis (p<0.01). Alcalase

treatment alone increased the IC50 value of kidney bean, black gram and

peanut to 4, 8.5 and 123 folds respectively. Sequential hydrolysis using

both alcalase and flavourzyme increased the IC50 of the kidney bean, black

gram and peanut to 25, 368 and 393 folds respectively further as compared

to single enzyme treatment. Basophil histamine release showed reduced

allergenicity of the hydrolyzed proteins. Significant reduction in the

biopotency of hydrolyzed protein of kidney bean, black gram and peanut

was observed in SPT where 6/13 kidney bean sensitive individuals, 8/13

black gram sensitive individuals and 9/15 peanut sensitive individuals were

found SPT negative to their respective hydrolysates.

CONCLUSIONS: Sequential hydrolysis by alcalase and flavourzyme

reduced the allergenicity of legume proteins. The method has potential to

reduce allergenicity of food extracts.

68 Comparative Analysis of IgE Binding Proteins in GM and Non-GM Rice Varieties Using Atopic Patients Sera

Chandni Mathur1, P. C Kathuria2, Shakuntala Lavasa3, Pushpa Dahiya4,

Anand Singh, PhD5; 1Institute of Genomics & Integrative Biology, Delhi,

India, 2National Allergy Centre, India, 3USLavasa Medical & Research

Center, India, 4M.D University, India, 5Institute of Genomics and Integra-

tive Biology, Delhi, India.

RATIONALE: Food allergy is recognized as IgE-mediated immune

response. Safety issues regarding foods derived from genetically modified

(GM) plants are central to their acceptance into the food supply and its

commercial production.

METHODS: Three GM rice varieties containing cry protein Cry 1ab, Cry

1ac and Cry 1c along with their native variety were selected for the present

investigation. All the seed powders were processed for protein extraction

and estimation. Extracts were subjected to SDS-PAGE for protein profil-

ing. Simulated Gastric Fluid Digestion (SGF) was also done for all seed

powders. IgE binding protein bands in rice were observed through

Immunoblot with sera of allergic patients. Bioinformatic analysis of above

transgenic 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

-14 bands in all varieties within a range of 9-81 kD.Native rice andGM rice

with Cry 1Ab subjected to SGF showed a very faint appearance of smear

around 9kD. Rice variety with Cry 1Ab had faint proteins around 27 and 47

kD. IgE binding protein bands observed in rice varieties were aggregating

around 37-47 kD. Comparative analysis across different databases yielded

that cry proteins used in GM Rice showed sequence identity of < 27 % .

CONCLUSIONS: It is suspected that no major detectable difference was

observed in GM and non-GM rice varieties.

69 Analysis of the Association Between Food Allergy andSensitization to Ltp and Profilin

Felicia Berroa1, M Jose Goikoetxea, PhD, MD2, Marta M.

Ferrer, MD, PhD, FAAAAI2, Paula Cabrera-Freitag, MD, PhD3, Gracia

Javaloyes, MD, PhD1, Maria L Sanz, MD, PhD1, Gabriel Gastaminza,

MD, PhD1; 1Department of Allergy, Clinica Universidad de Navarra,

Spain, 2Department of Allergy, Clinica Universidad de Navarra, Pam-

plona, Spain, 3Clinica Universidad de Navarra, Pamplona, Spain.

RATIONALE: LTP and profilin panallergens are associated with food

allergy (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 profilin.

METHODS: Retrospective analysis of 238 patients in whom skin prick

test (SPT) to palm profilin and peach LTP(ALK-Abell�o, Madrid, Spain)

was performed. Specific IgE rPhl p 12 and rPru p 3 were analyzed by

ImmunoCAP (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, 20

patients SS and 16 OAS and SS. Eighty-three patients had positve SPT to

profilin and/or LTP (42 profilin, 29 LTP and 12 both panallergens).Skin

sensitization to LTP was associated with: OAS (OR: 3.279 [1.595-6.741];

p50.001) 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 to

rPrup3 by ImmunoCAP was associated with OAS, only in those patients

suffering also from SS. ImmunoCAP (OR: 2,568 [1,254-5,258]; p50.008)

but not SPT (OR: 2.206[1.121-4.342]; p50.020) to profilin were associ-

ated with OAS without SS.

CONCLUSIONS: Having positive SPTand/or ImmunoCAP is associated

with having systemic symptoms with or without OAS. When OAS is

present without systemic symptoms is associated only with ImmunoCAP

to rPhl p 12.

70 Structure and Function of the Peanut Panallergen Ara h 8Barry K. Hurlburt, PhD1, Lesa Celeste, PhD2, Karolina A.

Majorek3, Jane McBride4, Soheila J. Maleki, PhD1, Wladek

Minor, PhD3, Maksymilian Chruszcz, PhD5; 1USDA-ARS-SRRC, New

Orleans, LA, 2university of south carolina, columbia, SC, 3University 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 responsible

for oral allergy syndrome between birch pollen and peanut. In addition, it

may also be a signaling molecule carrier. We sought to investigate these

notions with structural, and biochemical characterization.

METHODS: Recombinant Ara h 8 was expressed in E. coli and purified.

The structurewas determined using X-ray crystallography. Ligand binding

was tested by displacement of bound fluorescent 8-anilo-1-naphthalenesul-

fonic acid.

RESULTS: Purification by a combination of heat treatment, ammonium

sulfate precipitation and hydrophobic interaction chromatography yielded>95%pureAra h 8.The crystal structurewas solvedbymolecular replacement

and refined at 1.7�A resolution. The overall fold ofAra h 8was very similar to

Bet v 1, as well as Bet v 1 homologs Api g 1 (celery), Gly m 4 (soy), and Pru

av 1 (cherry). Physiologically-relevant ligands have been identified.

CONCLUSIONS: Our results support both the idea that Ara h 8 could be

contributing to oral allergy syndrome between birch pollen and peanut, and

the hypothesis that Bet v 1-like proteins could be transporters of signaling

molecules important for plant development.