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
Clinical and Experimental Allergy;2010 ,40: 896-904
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
Basic of allergen component
Clinical and Experimental Allergy;2010 ,40: 896-904
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
Basic of allergen component
Clinical and Experimental Allergy;2010 ,40: 896-904
• Production technique of allergen component can be
- biotechnological in “recombinant” from- “purified” from (original source)
Basic of allergen component
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
Important allergen families
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
Important allergen families
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.
Important allergen families
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.
Important allergen families
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
Egg (Gallus domesticus)
Clinical& experimental allergy; 2010: 40: 1442-1460
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
Egg (Gallus domesticus)
Clinical& experimental allergy; 2010: 40: 1442-1460
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
Egg (Gallus domesticus)
Clinical& experimental allergy; 2010: 40: 1442-1460
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)
Egg (Gallus domesticus)
Role in egg allergy remain unclear
Clinical& experimental allergy; 2010: 40: 1442-1460
• 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 -
Heated EW + Raw EW + Heated & raw EW -
JACI 2008;122:583-588
Heated EW +
Raw EW + Heated & raw EW -
Heated EW +
Heated EW +
Heated EW +
Raw EW +
Raw 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)
Whey
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)
Cow’s milk (Bos domesticus)
Curr Opin Allergy Clin Immunol 11: 216-221
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
Cow’s milk (Bos domesticus)
Curr Opin Allergy Clin Immunol 11: 216-221
• 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
(J Allergy Clin Immunol 2009;123:883-8.)
• Result
mo
Five of the 6 children had asthma
Severe reaction 15%
(J Allergy Clin Immunol 2009;123:883-8.)
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.
(J Allergy Clin Immunol 2009;123:883-8.)
fir
- 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.
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.
B. Ahrens, et.al, Clinical & Experimental Allergy, 2012 (42) 1630–1637.
18 mo
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)
Clinical& experimental allergy; 2010: 40: 1442-1460
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
Clinical& experimental allergy; 2010: 40: 1442-1460
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)
Clinical& experimental allergy; 2010: 40: 1442-1460
Tri a 19 (Ѡ-5 gliadin) - associated with risk of IgE-mediated reaction to wheat
- risk of wheat-dependent exercise induceanaphylaxis
Wheat (Triticium aestivum)
Clinical& experimental allergy; 2010: 40: 1442-1460
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)
Wheat (Triticium aestivum)
Clinical& experimental allergy; 2010: 40: 1442-1460
• 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
J Allergy Clin Immunol 2001;108:634-8
- 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
J Allergy Clin Immunol 2001;108:634-8
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)
Clinical& experimental allergy; 2010: 40: 1442-1460
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
Peanut (Arachis hypogaea)
Clinical& experimental allergy; 2010: 40: 1442-1460
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
Clinical& experimental allergy; 2010: 40: 1442-1460
Peanut (Arachis hypogaea)
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
Peanut (Arachis hypogaea)
Clinical& experimental allergy; 2010: 40: 1442-1460
• 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
J Allergy Clin Immunol 2010;125:191-7
J Allergy Clin Immunol 2010;125:191-7
J Allergy Clin Immunol 2010;125:191-7
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
Happy New year 2013
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