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212 Positive skin test and rast to peanut and almond in non-allergic individuals
VOLUME NUMBER
209
210
87 1, PART 2
Abstracts
ISOLATION OF PEANUT ALLERGENS USING WONOCLONAL ANTIBODIES. S.L. Hefle. M.S.. J.P. Folaert, @ S . F.S Chu. Ph.D.. R.K. Bush. N.D,, Madison, WI. Peanut allergy is one of the most common food allergies. The allergenic components of peanuts lie in protein or glycoprotein These allergens have not been fully fractions. characterized. Wonoclonal antibodies offer a stable, inexhaustible, and standard supply of reagent for quantitation and characterization We previously identified of peanut allergens. a series of proteins, with approximate molecular weights in the 15-25 kD range by SDS-polyacrylamide gel electrophoresis (PAGE). These peanut protein fractions showed allergenic activity in vivo when used to skin test peanut-sensitive patients, and in vitro by RAST and IgE immunoblotting studies. Wonoclonal antibodies were raised against these proteins, and used to arm affinity columns. Peanut proteins were affinity-purified from crude peanut extract using these columns and then characterized by SDS-PAGE and immunoblotting. Three bands with approximate molecular weights of 14 kD, 40 kD, and 65 kD were identified which bound IgE from peanut-sensitive patients. Further studies revealed that the monoclonal antibodies also recognize a previously described 65 kD concanavalin A-reactive peanut allergen (Barnett and Howden, Biochimica et Biophysics Acta, 1986, 882:97-105). These affinitypurified proteins will help us to more fully characterize the allergic response to peanuts.
211
PRODUCTION OF MURINE MONOCLONAL. (mAb) ANTIBODIES TO ARA h I, A 63.5 kD ALLERGEN IN PEANUTS. AW Bu&,&$D I,W . .
212
Wllhams. Little Rock, Arkansas. Our previous study has identified a 63.5kD allergen, Ara h I from peanuts,utilizing the serumof atients with atopic dermatitis and ltive doublet.lmd, lacebo-controlledfood chalp” engesto peanut. This Bergen isolated from crude extractsof Flonmner peanutswasused asan immuogento reduce mAbs. Spleencells from hyperimmunized f3ALB/c mice were fusedwith mousemyelomacells using standard rocedures. H bridoma cell lines were screenedFor secretionor mAb by ELISA using insolubilized Ara h I. Culture supematantsfrom thirty subcloneswere further screenedfor Ara h I binding activity by SDS-PAGE/immunoblot analysts.Four cloneswere then selectedfor
soybeanextracts. Current work is underwayto purify peanut allergensby mAb-affinity chromatography.These mAbs should allow for easierisolation of major aller ensand provide reagentsfor the future standardization of peanut extracts.
191
IDENTIFICATION OF A SECOND MAJOR PEANUT ALLERGEN IN PATIENTS WITH
Rock, AR. Peanutsare a common.causeof food hypersensitivityreactions. We usedthe seraof 10 patients who had atopic dermatitis and a sitive double-blind placebo-controlledfood chr lenge to peanut to investigatethe major allergensof eanut. Crude Flonumer extractswere fractionatedgy anion exchangechromat (limit buffer. O.oSI??&?i?~& 8?8ra~~t hundred microliters of each2.0 ml fraction was dotblotted onto nitrocellulose paper and IgE-binding activity assessedusing our serumpool to select allergen-containingfractions. A protein peak (OD 280) which eluted at 10% NaCl and demonstrated intense IgE-bindin wasfurther analyzedby twodimensionalSDS-dAGE/immunoblot analysis. The majori of this fraction is a protein which has a molecu7 ar weight of 17kD and a pl of 5.2. Sequencingdata from the N-terminus revealedthe following initial 9 amino acids: G-Q-G-(W)-E-L-QG-D. Basedon IgE-bindin activity and no known amino acid sequenceI3 entity to other allergens,we have designatedthis allergen Ara h II.
POSITIVE SKIN TEST AND MST TO PEANUT AND ALMOND IN NON-ALLERGIC INDIVIDUALS. J.A. Nordlee, B.S., and S.L. Taylor, Ph.D., Lincoln, Nebraska While screening non-peanut allergic individuals for negative control sera, three individuals reacted positively to skin prick test to peanuts. These individuals partake of peanuts on a regular basis with no ill effects. c Individual sera were analyzed by radioallergosorbant test (RAST) for peanut specific Peanut proteins were separated by sodium IgE. dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to nitrocellulose. Blots were incubated with individual sera and bound IgE was detected using Iodine-125 labelled anti-human IgE and autoradiography. The peanut specific RAST indicated that the three individuals with positive skin tests also have increased amounts (6X) of peanut specific IgE. The IgE in the serum of one individual bound to at least three protein bands as determined by autoradiography. Two of the peanut skin test positive individuals also had positive skin prick tests to almond, which both report eating with no ill effects. No binding was apparent on the blots, but both had elevated serum levels of almond specific IgE determined by RAST.
209
210
87 1, PART 2
Abstracts
ISOLATION OF PEANUT ALLERGENS USING WONOCLONAL ANTIBODIES. S.L. Hefle. M.S.. J.P. Folaert, @ S . F.S Chu. Ph.D.. R.K. Bush. N.D,, Madison, WI. Peanut allergy is one of the most common food allergies. The allergenic components of peanuts lie in protein or glycoprotein These allergens have not been fully fractions. characterized. Wonoclonal antibodies offer a stable, inexhaustible, and standard supply of reagent for quantitation and characterization We previously identified of peanut allergens. a series of proteins, with approximate molecular weights in the 15-25 kD range by SDS-polyacrylamide gel electrophoresis (PAGE). These peanut protein fractions showed allergenic activity in vivo when used to skin test peanut-sensitive patients, and in vitro by RAST and IgE immunoblotting studies. Wonoclonal antibodies were raised against these proteins, and used to arm affinity columns. Peanut proteins were affinity-purified from crude peanut extract using these columns and then characterized by SDS-PAGE and immunoblotting. Three bands with approximate molecular weights of 14 kD, 40 kD, and 65 kD were identified which bound IgE from peanut-sensitive patients. Further studies revealed that the monoclonal antibodies also recognize a previously described 65 kD concanavalin A-reactive peanut allergen (Barnett and Howden, Biochimica et Biophysics Acta, 1986, 882:97-105). These affinitypurified proteins will help us to more fully characterize the allergic response to peanuts.
211
PRODUCTION OF MURINE MONOCLONAL. (mAb) ANTIBODIES TO ARA h I, A 63.5 kD ALLERGEN IN PEANUTS. AW Bu&,&$D I,W . .
212
Wllhams. Little Rock, Arkansas. Our previous study has identified a 63.5kD allergen, Ara h I from peanuts,utilizing the serumof atients with atopic dermatitis and ltive doublet.lmd, lacebo-controlledfood chalp” engesto peanut. This Bergen isolated from crude extractsof Flonmner peanutswasused asan immuogento reduce mAbs. Spleencells from hyperimmunized f3ALB/c mice were fusedwith mousemyelomacells using standard rocedures. H bridoma cell lines were screenedFor secretionor mAb by ELISA using insolubilized Ara h I. Culture supematantsfrom thirty subcloneswere further screenedfor Ara h I binding activity by SDS-PAGE/immunoblot analysts.Four cloneswere then selectedfor
soybeanextracts. Current work is underwayto purify peanut allergensby mAb-affinity chromatography.These mAbs should allow for easierisolation of major aller ensand provide reagentsfor the future standardization of peanut extracts.
191
IDENTIFICATION OF A SECOND MAJOR PEANUT ALLERGEN IN PATIENTS WITH
Rock, AR. Peanutsare a common.causeof food hypersensitivityreactions. We usedthe seraof 10 patients who had atopic dermatitis and a sitive double-blind placebo-controlledfood chr lenge to peanut to investigatethe major allergensof eanut. Crude Flonumer extractswere fractionatedgy anion exchangechromat (limit buffer. O.oSI??&?i?~& 8?8ra~~t hundred microliters of each2.0 ml fraction was dotblotted onto nitrocellulose paper and IgE-binding activity assessedusing our serumpool to select allergen-containingfractions. A protein peak (OD 280) which eluted at 10% NaCl and demonstrated intense IgE-bindin wasfurther analyzedby twodimensionalSDS-dAGE/immunoblot analysis. The majori of this fraction is a protein which has a molecu7 ar weight of 17kD and a pl of 5.2. Sequencingdata from the N-terminus revealedthe following initial 9 amino acids: G-Q-G-(W)-E-L-QG-D. Basedon IgE-bindin activity and no known amino acid sequenceI3 entity to other allergens,we have designatedthis allergen Ara h II.
POSITIVE SKIN TEST AND MST TO PEANUT AND ALMOND IN NON-ALLERGIC INDIVIDUALS. J.A. Nordlee, B.S., and S.L. Taylor, Ph.D., Lincoln, Nebraska While screening non-peanut allergic individuals for negative control sera, three individuals reacted positively to skin prick test to peanuts. These individuals partake of peanuts on a regular basis with no ill effects. c Individual sera were analyzed by radioallergosorbant test (RAST) for peanut specific Peanut proteins were separated by sodium IgE. dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to nitrocellulose. Blots were incubated with individual sera and bound IgE was detected using Iodine-125 labelled anti-human IgE and autoradiography. The peanut specific RAST indicated that the three individuals with positive skin tests also have increased amounts (6X) of peanut specific IgE. The IgE in the serum of one individual bound to at least three protein bands as determined by autoradiography. Two of the peanut skin test positive individuals also had positive skin prick tests to almond, which both report eating with no ill effects. No binding was apparent on the blots, but both had elevated serum levels of almond specific IgE determined by RAST.