Component resolved diagnosis in food allergy

Sadudee Boonmee, MDDivision of Allergy and ImmunologyDepartment of PediatricsKing Chulalongkorn Memorial Hospital

Component Resolved Diagnosis in Food allergy

Topic outline

• Introduction • Important protein families• Allergen from animal origin (Egg, CM)• Allergen from plant origin (Soy, wheat

and peanut)

Currently available tests detect only sensitization, not clinical allergy, cannot predict prognosis and severity

Nature

Allergenic source

Nature

Allergenic source

contain a mixture of allergenic and non-allergenic molecules

Introduction • Specific IgE determined using allergen extracts as test

allergens have to 2 type of problem1. the difficulty of starndardizing the allergens used as substrates ( differ in allergenicity

variability of allergen source)

2. can not differentiated between primary sensitization and immunological cross-reactivity

Borres et.al,Pediatric Allergy and Immunol 2011;22:454-461.

Introduction

• Both skin prick testing and specific IgE blood testing employ heterogeneous protein preparations not always the best indicators of clinical reactivity

• Component testing designed to pinpoint the allergen source when cross-reactive allergen components are present.

Borres et.al,Pediatric Allergy and Immunol 2011;22:454-461.

Introduction

• Identifying whether the sensitization is genuine in primary (species-specific) or if it is due to cross-reactivity to similar protein structure may help to evaluate the risk of reaction on exposure to difference allergen source

Clinical and Experimental Allergy;2010 ,40: 896-904

1. Almost anything containing proteins can be an allergen source

- each allergen source contains many different allergenic protein (allergen component)- each allergen component commonly has several different epitopes - epitope is the actual three-dimensional binding site for an antibody

Basic of allergen component

2. Protein stability - allergen that stable to heat and digestion are more likely to cause severe reaction

- allergen that heat and digestion labile are more likely to be tolerateed or only cause mild/local symptom

3. Allergen component name - the first 3 letter of the genus, follow by the first letter of the species and the number indicating the order of allergen identification

- Latin designation of peanut is Arachis hypogaea thus Ara h 1 designated allergen number 1 from peanut

• Production technique of allergen component can be

- biotechnological in “recombinant” from- “purified” from (original source)

Important allergen families

• Most allergen component belong to a limited number of protein families

• IgE antibodies in the same protein family often cross-reactive, and classifying allergen component into protein families can answering question on cross-reactivity

LTP (non-specific Lipid Transfer Protein, nsLTP)- A protein stable to heat and digestion causing reactions also to cooked foods.- Often associated with systemic and more severe reactions in addition to OAS.- Often associated with allergic reactions to fruit and vegetables in southern Europe.

Storage proteins- Proteins found in seeds serving as source material during the growth of a new plant.- Often stable and heat-resistant proteins causing reactions also to cooked foods.

Plant allergen

Profilin -An actin-binding protein showing great homology and cross-reactivity even

between distant related species.- Recognized as a minor allergen in plants and plant related foods.- Seldom associated with clinical symptoms but may cause demonstrable or even severe reactions in a small minority of patients.

CCD- Cross-reactive carbohydrate determinants are seldom associated with clinical symptoms but may cause demonstrable or even severe reactions in a small

minority of patients.

PR-10 protein,Bet V1 homologue - A heat labile protein, cooked foods are often tolerated.

- Often associated with local symptoms such as oral allergy syndrome (OAS).- Often associated with allergic reactions to fruit and vegetables in northern Europe.

Lipocalin - Stable proteins and important allergens in animals

- Allergen components displaying limited cross-reactivity between species.

Parvalbumin- A major allergen in fish.- A marker for cross-reactivity among different species of fish and amphibians.- A protein stable to heat and digestion causing reactions also to

cooked foods.

Animal allergen

Tropomyosin- An actin-binding protein in muscle fibres.- A marker for cross-reactivity between crustaceans, mites and cockroach.

Serum albumin - A common protein present in different biological fluids and solids e.g. cow’s milk and beef, eggs and chicken.- Cross-reactions between albumins from different animal species are well known, for example between cat and dog and cat and pork.

Allergen of animal origin

Allergen component in egg white Gal d 1, 2, 3, 4

Egg (Gallus domesticus)

Clinical& experimental allergy; 2010: 40: 1442-1460

Gal d 5 alpha-livetin (serum chicken albumin)

Gal d 1(ovumucoid)- 10% of total egg white protein - stability against to heat and digestion by protease

(strong disulfide bonds) capacity to stimulate a specific immune response

- sIgE Ab to Gal d 1 helpful in prognosis and diagnosis of egg allergy

- High concentration of ovomucoid-sIgE associated with persistent egg allergy

(raw or cooked egg )- Low concentration of ovomucoid-sIgE

associated with tolerance to heated egg

Gal d 2 (ovalbumin)- heat-labile, IgE - binging epitopes on OVA might be detroyed after heating less allergenic- children who have sIgE primary to OVA likely to tolerate heated egg

Gal d 3(ovotransferrin)- nonheme iron-binding- heat-labile allergen - sIgE Ab to ovotransferrin in Dx of egg

allergy not been determined Gal d 4 (lysozyme)- lysozyme commomly used as a food

preservative(antibacterial) - egg allergic pt. who sensitized to lysozyme may react when exposured to such product

Allergenic component in egg yolk : Gal d 5Gal d 5 (alpha-livetin) = chicken serum albumin- bird-egg syndrome Vitellenin(apovitellenin I)apoprotein B (apovitellenin VI)

Role in egg allergy remain unclear

• Objective: to evaluate the clinical usefulness and added diagnostic value of measurements of IgE antibodies to egg white, ovalbumin, and ovomucoid in children with egg allergy.

• Methods: 108 children age 14 mo – 13 y (median age, 34.5 mo) with suspected egg allergy underwent double-blind, placebo controlled food challenges with raw and heated egg.

• The outcomes of the challenges were related to the serum

concentration of specific IgE antibodies and total IgE by using ImmunoCAP

JACI 2008;122:583-588

• Pt. devided into 3 group ( immediated reactions to OFC)- Group A (n=38) : positive challenge for heated egg white- Group B (n=29) : tolerated heated egg white, positive

challenge to raw egg white - Group C (n=41) : tolerated both heated and raw egg white

• Serum of all pt. obtain before food challenge- sIgE egg white, sIgE to ovalbumin, sIgE to ovomucoid, total IgE ImmunoCAP

JACI 2008;122:583-588

Results

JACI 2008;122:583-588

Heated EW + Raw EW + Heated & raw EW -

JACI 2008;122:583-588

Heated EW +

Raw EW + Heated & raw EW -

Heated EW +

Raw EW +

Heated & raw EW - Heated & raw EW -

Heated & raw EW -

children allergic to raw egg white(ie, groups A and B vs group C; A)

children allergic to heated egg white (ie, group A vs groups B and C; B).

sIgE Ab to egg white well in diagnosis of reactions to raw egg white- positive decision point was 7.4 kUA/L- negative decision point was 0.6 kUA/L

sIgE Ab to ovomucoid showed the best results in patients who react to heated egg white- positive decision point was 10.8 kUA/L- negative decision point was 1.2 kUA/L

• Quantification of sIgE to OVM could be useful in guiding physician in decision whether to perform a challenge or not

• OVM > 11 kUA/L high risk of reaction to heated and raw egg• OVM < 1 kUA/L low risk of reaction to heated egg (pt. may

react to raw egg)

Clinical implications: Measurement of specific IgE antibodies to egg white and ovomucoid might be helpful in guiding evaluations of children for allergy to raw egg or heated egg (eg, in cakes/cookies).

JACI 2008;122:583-588

Cow’s milk (Bos domesticus)

Caseins

Curr Opin Allergy Clin Immunol 11: 216-221

Caseins= 80% of total proteinβ lactoglobulin (β-LG) not present in human milk

Major allergen in milk

α-lactoglobulin (Bos d 4)- Functional subunit of whey lactose syntase- controversial role in milk sensitization - 80% of CMPA react to Bos d 4β-lactoglobulin (Bos d 5)

- most abundant whey protein - Pt. have allergic reaction to β-lactoglobulin 13-76%

Bovine serum albumin (BSA) (Bos d 6)- cow’s milk and beef allergy - CMPA pt. have positive to BSA 0-88% (only 20% have clinical reaction)

Bovine immunoglobulins (Bos d 7)- in blood, tissue fluid, secretion - seldom cause clinical symptom in CMPA

Caseins (Bos d 8) - αs1-caseins, αs2-caseins, β-caseins, κ-caseins

- polysensitization to many caseins fraction usually observed (closely epitope or cross-sensitization )

- αs1-caseins major allergen inducing strong immediated or delayed allergic reactions

• Objective: to calculate the frequency of accidental

exposure reactions in children allergic to cow’s milk during a 12-month period

: to analyze clinical characteristics andcircumstances surrounding the reactions

: to identify risk factors for severe reactions

(J Allergy Clin Immunol 2009;123:883-8.)

• Methods: 88 children allergic to cow’s milk (44 boys; median age,

32.5 months)

: cross-sectional study at age >= 18 mo : Systematized questionnaire about accidental

exposure (reactions were classified as mild, moderate, and severe)

: sIgE to cow’s milk & casein titers were determined periodically as clinical indicated in F/U visit

• Result

Five of the 6 children had asthma

Severe reaction 15%

P, Percentile.Mann-Whitney U test: #/{P 5 .044; #/kP < .01; /P 5 .044; /**P < .01.*P value: comparison between severe AARs versus mild or moderate AARs or no reaction.Fisher test.Mann-Whitney U test.§x2 Test.

Conclusion : Reactions to accidental exposure are frequent inchildren with cow’s milk allergy. The proportion of severereactions was 15%. The risk factors for severe reactions included very high levels of specific IgE to cow’s milk and casein and asthma.

- Retrospective studies of 52 patients (3 to 114 months) with proven CMA by DBPCFC

- All Pt were rechallenge at least once , some were rechallenge 2 or 3 time

- 32 (61.5%) patients became tolerant in the analysed time period.

- Serum was collected at all time point prior to OFC analysed for

B. Ahrens, et.al, Clinical & Experimental Allergy, 2012 (42) 1630–1637.

• Result sIgE CM

18 mo 33 mo

Children with initial sIgE CM below 5.0 kU/L 4 time likely to tolerant

B. Ahrens, et.al, Clinical & Experimental Allergy, 2012 (42) 1630–1637.

Children with initial low (<0.3ISU/L) sIgE to α lactalbumin, β lactoglobulin(Bos d 5.0102), κ casein and αs1- casein 7.2, 2.7, 3.8 and 2.7 time likely to tolerant respectively

• Conclusion• Children became tolerant earlier if their specific IgE-antibody

levels against the α-lactalbumin, and β-lactoglobulin(Bos d5.0102), αs1- casein and, κ-casein were low

• CM-specific IgE is a good prognostic marker for persistence of CM

Allergen of plant origin

Allergen component in soy : Gly m 4, 5 and 6 Gly m 4- PR-10 protein family (Bet v 1 homologue) - pollen-associated soy allergy mainly discribed in adult with mild symptoms (primary sensitization to birch pollen)- cross-reactive to Ara h 8- In Europe approximately 2/3 of pt. allergic to soy are allergic to peanut

Soy (Glycine max)

Gly m 5 and 6- most important in primary sensitization to soy protein (via GI tract) esp. in children - sensitization rate to Gly m 5 36%Gly m 6 43% Gly m 5 and Gly m 6 are diagnostic marker for

severe allergic reaction to soy

Soy (Glycine max)

In soy allergic pt.

Paper soy2paper

• Aim : to identify relevant soybean allergens and correlate the IgE-binding pattern to clinical characteristics in European patients with confirmed soy allergy

• Methods : The IgE reactivity in 30 sera from subjects (Switzerland, Denmark and Italy)— with a positive double-blind, placebo-controlled soybean challenge (n = 25)

or a convincing history of anaphylaxis to soy (n = 5) was analyzed by ELISA or CAP-FEIA

J Allergy Clin Immunol 2009;123:452-8.

The 2 soybean major storage proteins- Gly m 5 (β-conglycinin : 3 subunits): α (67kd): α ‘ (71 kd):β (50 kd))- Gly m 6 (glycinin : 5 different subunits): G1 (A1aB1b, 53.6 kd): G2 (A2B1a, 52.4 kd): G3 (A1bB2, 52.2 kd): G4 (A5A4B3, 61.2 kd): G5 (A3B4, 55.4 kd)

• IgE reactivities major storage Protein (8 subunits) of the proteins were correlated to the clinical characteristics of our patient group to investigate the potential use as biomarkers for severe allergic reactions to soybean. J Allergy Clin Immunol 2009;123:452-8.

Clinical implications: Component-resolved diagnosis with purified Glym 5 and Glym 6 is potentially applicable for identifying subjects at risk of experiencing severe soy-related allergic reactions.

J Allergy Clin Immunol 2009;123:452-8.

Gly m 5 +ve = 13/30 (43%)Gly m 6 +ve = 11/30 (37%)Gly m 5 ot Gly m 6 +ve =16/30(53%)

Tri aA_TI, Tri a 18, Tri a gliadin, Tri a 19 (Ѡ- 5 gliadin) and high molecular weigh glutenin

Tri a 14 - Lipid transfer protein - heat resistance and lack of cross-reactivity to pollen - major allergen in pt. living in southern Europe and significant in baker’s asthma

Tri a 18 - Hev b 6 homologue (latex component)

Wheat (Triticium aestivum)

Tri a 19 (Ѡ-5 gliadin) - associated with risk of IgE-mediated reaction to wheat

- risk of wheat-dependent exercise induceanaphylaxis

Tri aA_TI (alpha-amylase/tyrpsin inhibitor)- wheat allergen in raw and cooked food - play role in wheat-depedent exercise induce

anaphylaxis

High molecular weigh glutenin- associated with wheat-dependent exercise

induce anaphylaxis

Tri a gliadin - primary wheat sensitization (low-risk of pollen cross-reactivity)

• Aim : to examine whether ω-5 gliadin is a clinically relevant allergen in children with hypersensitivity reactions to ingested wheat.

• Method : Sera were obtained from 40 children (mean age, 2.5 years; range, 0.7-8.2 years) with suspected wheat allergy (AD a/o GI a/o

respiratory symptoms : Wheat allergy Dx with open or DBPC oral wheat challenge.

: Wheat ω-5 gliadin was purified by reversed-phase chromatography,and serum IgE antibodies to ω-5 gliadin were measured by ELISA

: In vivo reactivity was studied by SPT. : Control sera were obtained from 22 children with no

evidence of food allergies

J Allergy Clin Immunol 2001;108:634-8

• Result

Immediated

Delayed

- 16/19(84%) detected IgE to Ѡ-5 gliadin

- 6/7 SPT +ve to Ѡ-5 gliadin

- not detected IgE to Ѡ-5 gliadin- 2/2 SPT –ve to Ѡ-5 gliadin

Conclusion: The results of this study show that ω-5 gliadin is a significant allergen in young children with immediate allergic reactions to ingested wheat. IgE testing with ω-5 gliadin could be used to reduce the need for oral wheat challenges in children.

Morita,et al. Allergology International.2009;58:493-498

- most common food associated with fatal allergic reaction in western country, the prevalence also increase in Asia

- 13 peanut allergen have been identified - 5 peanut component are clinically

relevant and available

Ara h1, 2, 3, 6, 8, and 9

Peanut (Arachis hypogaea)

Ara h 1, 2, and 3 - major peanut allergen (peanut seed storage protein)

- associated with primary sensitization - pt. with peanut allergy are sensitized to Ara h 1 35-95%Ara h 2 75-100%Ara h 3 20-55% - stable to heat and digestion risk of reaction to

cooked or roast food

Ara h 1 - cross-reactive to nut and legume [lentil,pea, and soy(Gly m 5)]

Ara h 2- marker for primary sensitization to peanut and risk marker for severe allergic reaction- cross-reactive to lupine and tree nut (eg. almond and brazil nut)

Ara h 3 - cross-reactive to soybean, pea, and tree nuts

Ara h 6- high homology to Ara h 2Ara h 8 - PR-10, Bet V 1 homologue- heat-labile protein cooked food often tolerated - primary sensitization through pollen : birch,cedar- cross-reactive to lupine and soy (Gly m 4) Ara h 9 - Lipid transfer protein (LTP)- associated with severe OAS and systemic reaction - primary sensitization probably due to primary sensitization to peach or other LTP-containing fruits

• Aim - To establish by oral food challenge the proportion of children with clinical peanut allergy among those

considered peanut-sensitized by using skin prick tests and/or IgE measurement

- To investigate whether component-resolved diagnostics using microarray could differentiate peanut allergy from tolerance.

• Method - The Manchester Asthma and Allergy study

unselected population based birth cohort - participants recruited prenatally and F/U prospectively at age 1, 3, 5 and 8 yr ( questionnaire, SPT, sIgE )J Allergy Clin Immunol 2010;125:191-7

85 were sensitized to peanut

13 OFC

Green : peanut tolerantYellow : peanut allergy

- Peanut allergy fold change = 6.06 expression level 66.7 time higher than negative control - Peanut tolerant fold change = 0.28 expression level 1.2 time higher than negative control

Clinical implications: Measurement of IgE response to major peanut allergen Ara h 2 is more useful in predicting clinical peanut allergy than currently used skin or blood tests based on whole extract.

Advantage of CRD

• Quantifying IgE antibodies to a single allergen protein component rather than a heterogeneous allergen preparation

• Providing a unique component-specific IgE antibody profile of the patient for personalized medical care

• Supporting a molecular-level understanding of the patient’s individual constellation of symptoms

Advantage of CRD

• Better characterize the patient’s sensitization patterns• Better understand the clinical risk for reactions in a patient• Determine the presence of cross-reactivity between allergens• Facilitate food challenge testing with increased knowledge of what to expect• Select appropriate patients for specific immunotherapy and customize treatment

Advantage of CRD

Allergen Components as severity markers

Peanut Ara h 1, 2 and 3

Soy Gly m 5 and 6

Wheat Omega-5-gliadin

Milk Bos d 8 (casein)

Egg Gal d 1 (ovomucoid)

Important allergen

Ara h 1, 2, 3, 8 and 9 (peanut)• IgE abs to Ara h 1, 2, 3 and 9 (LTP) are associated with peanut reactions, which in many cases can be severe.• IgE abs to Ara h 8 (PR-10) are usually associated with milder, local symptoms such as OAS, originting from birch sensitzation.

Ovumucoid (egg white)• IgE abs to ovomucoid are associated with persistent egg allergy and usually neither raw or cooked is tolerated.

Omega-5 gliadin (wheat)• IgE abs to omega-5 gliadin (Tri a 19) in adults are associated with a risk of exercise- or NSAIDs-induced reactions in connection with wheat ingestion.• IgE abs to omega-5 gliadin in children are associated with a risk of immediate reactions to wheat.

Gly m 4,5 and 6 (soy)• Gly m 5 and 6 are associated with clinical reactions to soy. Gly m 5 - Ara h 1 and Gly m 6 - Ara h 3 share homologoues structures.• IgE abs to Gly m 4 (PR-10) are usually associated with milder, local symptoms such as OAS, originating from birch sensitzation.

Important allergen

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Component resolved diagnosis in food allergy

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