Laboratory Diagnosis of Human Allergic Disease R.G. Hamilton, Ph.D. D.ABMLI Professor of Medicine and Pathology Johns Hopkins University School of Medicine Baltimore, MD 2018 June

Dynamic Factors That Alter the Degree of Allergic Sensitization allergen Patient Factors age (child vs adult), gender, race , social economic status, family atopy history-genetic predisposition, immune status Environmental Factors allergen source (complexity-concentration), duration and route of exposure, environment, pet owner-ship, smoking continued allergenic challenge IgE Antibody Quantity/Quality Changes Concentration (kUa/L) Affinity (tightness of binding) Ka/Kd Clonality (epitope specificity) Specific Activity (specific IgE to total IgE ratio)

Diagnostic Algorithm for Allergic Disease

Clinical History Predictors of Atopy (IgE+ sensitization) Hoppin JA et al,d NHANES 2005-2006 History Predictors of Atopy: Am J Epidemiol 2011; 173:544-552 8334 Hx and IgE antibody-17 specificities 44% IgE Ab Positive=Atopic poor diagnostic sensitivity (2-42%) better diagnostic specificity (72-98%) eczema: PPV=50%, NPV=57% hayfever: PPV=72%, NPV=65% Clinical History is unreliable as a sole indicator of atopy (allergy) Hay fever Allergy Eczema Rhinitis Itchy rash

Diagnostic Algorithm for Allergic Disease Allergen IgE Antibody positivity indicates sensitization, not allergic disease!

Diagnostic Algorithm for Allergic Disease Allergen Allergen

Sensitization ≠ Allergic Disease The clinical relevance of allergic sensitization (positive allergen-specific IgE independent of method (skin test/serology) or diagnostic allergen source (extracts or molecules) can only be determined by the physician-based on the individual’s clinical history and not by the test alone

Terminology allergen – an immunogen (molecule) that when introduced into a host elicits the formation of IgE antibodies. Inhaled allergens (n=512) (41G+62W+122T+22DM+101M+80E+18O+7P+59 Misc) Ingested (food) allergens (n=363) Injected (stinging insect) allergens (n=48) Injected/Ingested (drug) allergens (n=47) Total: 970 individual allergen specificities

Diagnosis of Human Allergic Disease Allergen Extract Based Skin Testing Provocation testing 1880 2013 In-vivo (extract based skin and allergen challenge) testing

Allergen Extracts Used For Diagnosis and Immunotherapy in USA 19 standardized allergen extracts controlled for potency and stability D. farinae, D. pteronyssinus, cat hair, cat pelt, short ragweed, Hymenoptera (HBV, PWV, YJV, YHV, WFHV, mixed vespid venom), Grass pollens (Bermuda, red top, June, perennial rye, orchard timothy, meadow fescue, sweet vernal) 1269 non-standardized allergen extracts: no defined potency, composition, stability (expiration date?)

FDA Allergen Efficacy Review Panel 1, 21 CFR 601.25 (1974-1979): 773 extracts Panel 2, 21 CFR 601.26 (1982-1983): 752 extracts Internal FDA Review 2004-2006: >1500 allergen extracts 1269 non-standardized extracts: Animal: 28, Molds 180, Dusts: 6, Plants 16, Foods 277, Pollens: 708, Insects: 34 Table 1: Use in Diagnosis and Treatment Addressed in Literature (480) Table 2: Food: Use in Diagnosis Addressed in Literature (134) Table 3: Non-food: Use in Diagnosis Addressed in Literature (73) Table 4: Minimal/no literature related to Diagnosis or Treatment (566) Table 5: Potential Safety Issues (16): e.g. house dust, monkey pelt Slater JE…Rabin RL: The US FDA review of the safety and effectiveness of non-standardized allergen extracts JACI 129:1014-1019, 2012

Diagnosis of Human Allergic Disease Allergen Extract Based Serological Assays Characterisation of IgE Provocation testing RAST* 1880 1967 2013 In-vitro (extract based serological IgE Antibody Assay) testing In-vivo (extract based skin and allergen challenge) testing Wide L, Bennich H, Johansson SGO: Diagnosis by an in vitro test for allergen specific IgE antibodies. Lancet 2:1105-1109, 1967

Principal Analytes Measured in Diagnostic Allergy Laboratories Allergen-specific IgE (over 200 allergen specificities) Pollen (weeds, grasses, trees), Epidermals, Dust Mites, Molds, Foods, Venoms, Occupational allergens, Drugs Total Serum IgE (Xolair: anti-IgE; ABPA) Mast Cell Tryptase (indicator of anaphylaxis) Precipitating IgG Antibody (Hypersensitivity Pneumonitis) Eosinophil Cationic Protein (eosinophil activation marker)

IgE-Antibody Assay Chemistry Calibration: WHO IgE Standard Controls and Patient Test Sera Total-IgE Allergen-specific IgE 2. Anti-IgE IgE Allergen- (Mix) 1. Anti-IgE solid phase solid phase

Evolution of Allergen-Specific IgE Assay Technology Clinically-Used IgE Antibody Assays in North America ImmunoCAP (250, 1000): Phadia (Pharmacia, Jan 06)-76% Immulite 2000/2500: Siemens Medical Solutions Diagnostics (DPC-Jan 07)-21% HYTEC-288: Hycor Biomedical (June 07)- 3% New IgE Antibody Assays Immuno Solid Phase Allergy Chip (ISAC): Phadia/VBC Genomics – 20 ml of serum - 143 allergenic components ImmunoCAP Rapid: Lateral Flow (Over the Counter) IgE Antibody Assay-finger stick-10 aeroallergen specificities

FDA-Cleared IgE Antibody Single-plex Autoanalyzers ImmunoCAP-Phadia- ThermoFisher Scientific Immulite-Siemens Immulite Siemens Basic reagents and chemistries are similar * Allergen on solid phase binds antibody * Buffer wash to remove unbound protein * Enzyme anti-IgE detects bound IgE *All assays report in similar units with comparable analytical sensitivities of 0.1 kUa/L All assays principally use allergen extracts; Limited use of purified allergens (e.g., insulin) HYTEC Hycor Biomedical

Three Studies Verify Differential IgE Antibody Assay Results Wood RA, Segall N, Ahlstedt S, Williams PB. Accuracy of IgE antibody laboratory results. Ann Allergy Asthma Immunol. 2007;99:34-41. Wang J, Godbold JH, Sampson HA. Correlation of serum allergy (IgE) tests performed by different assay systems. J Allergy Clin Immunol. 2008 ;121:1219-24. Hamilton RG. Proficiency Survey Based Evaluation of Clinical Total and Allergen-Specific IgE Assay Performance. Arch Path Lab Med, 2010

IgE anti-Peanut Measurements by ImmunoCAP and IMMULITE

Allergen Extract Based Diagnostic IgE Antibody Tests Inhalants Tree Pollen Weed Pollen Grass Pollen Epidermals Insects Mites Molds Injected Venoms-Drugs Ingestants Foods 21

Advantages of Allergen Extract use in IgE Antibody Assays Practical issue: extracts of biological materials are easier to prepare Physiological extracts (in theory) contain the most comprehensive profile of allergens of clinical relevance achievable for that specificity

Problems with Allergen Extract Use in IgE Antibody Assays - Difficult to standardize complex allergen extracts due to natural variability of allergen sources Assays using extracts detect different populations of IgE antibody Cannot differentiate between primary sensitization and immunological cross-reactivity Cannot predict risk or identify prognostically signi- ficant sensitizations

Diagnosis of Human Allergic Disease Evolution from Allergen Extracts to Components Characterisation of IgE First allergens cloned Diagnostic recombinant allergen panels Provocation testing RAST* 1880 1967 1988-91 1995-1999 2013 In-vivo testing In-vitro testing Component-resolved diagnosis

Terminology allergen molecule: individual native or recombinant allergen with unique MW, pI, carbohydrate composition, nucleotide and/or amino acid sequence and reactivity to a monospecific antibody. Its allergenic property needs to be verified by binding to IgE antibody and/or induce a positive skin test or histamine release from basophils from at least 50% or more patients who are clinically allergic to the allergen specificity.

Application of purified component allergens-into IgE antibody assays Purified molecular allergens (recombinant [r] and native [n]) Peanut (Arachis hypogaea) Ara h 2 F13 Genus Species # 26

Common Properties of Allergens Stable to processing (heat) and digestion (multiple cysteine linkages) Pervasive – Abundant in nature Tend to form aggregates/polymers Many are defense related Interactions with lipid structures Actions interfere with common pathways Not every member of a protein family is allergenic or crossreactive (structure vs protein sequence)

Allergen Extracts are Imperfect but not Obsolete Complex Allergen Extract Recombinant molecular allergens

Allergen Component Related Knowledge Levels Components in a Single Allergen Extract Specificity

Allergen Component Related Knowledge Levels Components in a Single Allergen Extract Specificity: Cat: (Felis domesticus) Fel d 1 (uteroglobulin) Fel d 2 (albumin) Fel d 3 cystatin Fel d 4 lipocalin Fel d 5 IgA Fel d 6 IgM Fel d 7 Lipocalin Fel d 8 Laterin (sweat)

Early (FDA cleared) Components available on Singleplex Autoanalyzers * Food: (cow’s milk) nBos d 4,5,6 (a-lactoabumin; b-lactoglobulin ; casein) * Food: (chicken egg) n Gal d 1,2 (ovamucoid, ovalbumin) * Occupational (a-amylase) nAsp o 21 (Aspergillus oryzae) Baur X et al. Allergic asthma caused by exposure to bacterial alpha-amylase termamyl® Am J Ind Med. 2012 epub Pytelková J et al, Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro. BMC Biochem. 13:3, 2012

Allergen Component Related Knowledge Levels Components in a Single Allergen Extract Specificity 2. Components in Allergen Families With Defined Biological Functions

Protein/Allergen Families PFAM + SDAP (Structural Database of Allergenic Proteins) = AllFam Database There are circa 11,912 protein families Allergenic proteins confined to 236 PFAM’s (2% of total possible) 31 of these families contain multiple allergenic proteins (homologues, orthologues) Allergens comprises a small fraction of protein families with particular biological structures and functions

Established Molecular Allergen Families Tropomyosin Serum Albumin Profilin Carbohydrate Cross-reactive Determinants Pathogenesis Related Proteins: PR10 Family Non-specific Lipid Transfer Proteins (nsLTP) Polcalcin (calcium binding proteins) Lipocalin (fatty acid transport molecules) Parvalbumin Storage proteins

Part A: Molecular Allergology: General Concepts allergens, diagnostic applications, serological and cellular assays, allergen families Part B: Practical Guide to IgE Molecular Assays in Allergic Diseases tree pollen, grass pollen, weed pollen, dust mite, cockroach, pet epidermal, ABPA, atopic dermatitis, Hymenoptera venom (bee, vespid), occupational (latex, lab animals), foods (cow’s milk, chicken egg, fish, crustacean-mollusks, mammalian meat, fruit-vegetable, wheat, soy, peanut, tree nut and seed) Part C: Cross-reactive Molecules and their Clinical Relevance profilins, PR10-like (Bet v 1), non-specific lipid transfer proteins (nsLTP), albumins, tropomyosins, polcalcins, lipocalins, parvalbumins Matricardi P, Ollert M, Kleine-Tebbe J, Hoffman HJ, Valenta R, Editors, 2016

Allergen Families Tropomyosin: Actin-binding sites; muscle protein that regulates actin mechanics in muscle contraction, stable to heat and digestion; OAS and severe/systemic reactions: Anisakis-Ani s 3; Cockroach-Bla g 7; Dust mite-Der p 10; Shrimp Pen m 1 Profilin: An actin-binding protein in pollens and foods; involved in the dynamic turnover and restructuring of the actin cytoskeleton; sensitive to heat and digestion; Birch -Bet v 2; Natural Rubber Latex -Hev b 8; Mercury-Mer a 1; Timothy grass -Phl p 12

Allergen Component Related Knowledge Levels Ribonuclease: Pathogenesis Related Protein Group 10: Bet v 1 (birch), Cor a 1 (hazelnut), Mal d 1 (apple), Pru p 1 (peach), Gly m 4 (soybean), Ara h 8 (peanut), Act d 8 (kiwi), Api g 1 (celery), Pru av 1 (cherry) Birch tree Bet v 1 Birch Pollen

Symptoms Suggesting Sensitization Pattern Oropharyngeal symptoms after eating cherries, raw apples, hazelnuts, carrots, celery and/or soy AND symptoms of allergic rhinoconjunctivitis during the birch pollen season: Suggestive for the presence of IgE to major birch pollen allergen Bet v 1-specific (primary inhalant sensitization) Birch Cherry Celery Peanut

Pathogenesis Related Proteins: PR10 Family (Bet v 1 homologues)-Ribonuclease Treudler R., Update on in vitro allergy diagnostics. JDDG, 10:89-99, 2012

Component Resolved Diagnostics (CRD) Component resolved diagnosis may provide enhanced diagnostic value Traditional diagnostics CRD distinguish primary sensitization from cross-reactivity (Figure courtesy of Dr. Robert Wood)

Allergen Families  Polcalcin: Calcium binding protein; cross-reactivity among pollens: Timothy grass-Phl p 7; Birch-Bet v 4 Lipocalins: Fatty acid transport molecules; stable proteins in animals; cross-reactivity among species [Can f 2/Fel d4] Dog:-Can f 1,2,4,6; Cat-Fel d 4,7, Mouse-Mus m 1; Horse-equ c 1,2; Cow-Bos d 2,5; Rat-Rat n 1; D. Farinae-Der f 13; Cockroach-Bla g 4 Parvalbumins: Calcium buffering and transport; stable to heat and digestion; Often associated with systemic reactions; marker of cross-reactivity among fish and amphibians Cod-Gad c 1; Herring Clu h 1; Carp-Cyp c 1; Tuna-Kat p 1; Trout –Onc m 1; Flounder-Mackerel-Par o 1; Shrimp-Cra c 4,6; Lobster-Hom a 6; Frog-Ran e 1,2; Crayfish-Pen i 4

Allergen Families Non-specific Lipid Transfer Proteins: plant proteins in seeds/nuts that shuttle phospholipids /fatty acids between cell membranes; stable to heat/digestion: OAS and systemic reactions Peanut-Ara h 9; Hazelnut-Cor a 8; Walnut -Jug r 3; Peach-Pru p 3; Mugwort -Art v 3; Olive Pollen-Ole e 7; Plane tree -Pla a 3 Storage Proteins: In nuts and seeds-source of material for growth of new plants; stable to heat and digestion; often associated with systemic and more severe reactions Peanut-Ara h 1, 2, 3,6; Hazelnut-Cor a 9; Walnut-Jug r 1,2, soybean-Gly m 5,6

Allergen Families Carbohydrate Cross-reactive Determinants:  Serum Albumin: Protein that functions to transport fats and fatty acids to muscle tissue; fairly sensitive to heat and digestion : Cow-Bos d 6; Dog-Can f 3; Horse-Equ c 3 Cat -Fel d 2 Carbohydrate Cross-reactive Determinants: Rarely cause reactions; can produce positive in vitro test results to CCD containing allergens from pollens, plants, insects and venoms Bromelain (pineapple)-MUXF3: sensitization via pollen, insect venom Galactose-a-1,3 galactose- sensitization via tick bites, parasites?

Cross-reactive Allergen Families Tropomyosin: Actin-binding muscle protein that regulates actin mechanics in muscle contraction: Anisakis-Ani s 3; Cockroach-Bla g 7; Dust mite-Der p 10; Shrimp Pen m 1 Serum Albumin: Protein that functions to transport fats and fatty acids to muscle tissue : Cow-Bos d 6; Dog-Can f 3; Horse-Equ c 3 Cat -Fel d 2 Non-specific Lipid Transfer Proteins: plant proteins that shuttle phospholipids /fatty acids between cell membranes Peanut-Ara h 9; Hazelnut-Cor a 8; Walnut -Jug r 3; Peach-Pru p 3; Mugwort -Art v 3; Olive Pollen-Ole e 7; Plane tree -Pla a 3

Cross-reactive Allergen Families Profilin: An actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton Birch -Bet v 2; Natural Rubber Latex -Hev b 8; Mercury-Mer a 1; Timothy grass -Phl p 12   Thaumatin-Like Protein Green Kiwi - Act d 2 Carbohydrate Cross-reactive Determinants: Bromelain (pineapple)-MUXF3: sensitization via pollen, insect venom Galactose-a-1,3 galactose- sensitization via tick bites, parasites?

Clinical Significance of Allergen Families Clinical Significance of Allergen relates to stability to heat and gastric digestion Labile protein (low amount)---------------------------Stable protein (high amount) Profilin PR-10 LTP Storage Proteins Lipocalins Parvalbumin Tropomyosin Increasing risk to cause severe symptoms and reactions---------------------------

Strengths of Allergen Component based IgE Measurements Optimizing analytical sensitivity by replacement or supplementation of relevant allergens in test extracts (Hevea brasiliensis-Hev b 5; hazelnut-Cor a 1) Standardization of allergen extracts using individual allergens More precisely defining sensitization profiles for complex patients sensitized to many allergen groups Detection of cross-reactions and predictive risk factors (e.g., peanut)

Top Down Diagnostic Algorithm for Allergic Disease suspected allergy individual history (clinical symptoms?) examination (clinical findings?) A extract-based sensitization test(s) with interpretation SPT and/or IgE test and/or BAT (with extracts) B1 2nd IgE testing (selected molecules, CRD) molecular-based IgE-testing B2 interpretation aggreement with history? clinically relevant? B3 C interpretation (optional challenge) certain uncertain Kleine-Tebbe et al EAACI 2015 therapeutic consequences (i.e. allergen avoidance, allergen immunotherapy) D

Peanut

Utility of Component specific IgE Measurements in Peanut Allergy Allergen-specific IgE has limited diagnostic specificity for food allergy Specificity of a positive test for peanut extract specific IgE is less than 50% in children Oral food challenge is expensive, time consuming, and potential harmful -sensitized people can have measurable allergen-specific IgE and manifest no clinically-evident symptoms upon exposure to allergen -some allergenic components may be principally responsible for the induction of clinical reactions -whole peanut extracts contain clinically less important allergenic proteins that cross-react with prevalent aero-allergens such as Bet v 1 -peanut extract specific IgE antibody levels display a good diagnostic sensitivity for detecting sensitization, the diagnostic specificity of a positive test at a 0.35 kUa/L cut-off is less than 50% in children eleven allergenic peanut proteins have been identified, although the clinical significance of the two most recently characterized (Ara h 10 & Ara h 11)[pons l 2002, Allergy, pons l 2005] has not been established. Ara h 1, 2, 3, 4, 6 and 7 are seed storage proteins. Ara h 3 and 4 are structurally related, as are Ara h 2, Ara h 6 and Ara h 7.[Lauer I 2009] Ara h9 is a member of the non-specific lipid transfer protein family (nsLTPs). The seed storage proteins and the lipid transfer proteins are heat and digestion stable and thus they tend to be more commonly associated with clinical reactivity. In contrast, Ara h8 is a member of the pathogenesis-related 10 (PR-10) family which includes allergens such as Bet v 1 from Birch that are heat-labile and highly cross-reactive.[Sastre] Ara h5 is another heat-labile protein of the profillin family that is cross-reactive with Bet v 2 (Birch) and Phl p 12 (Grass), and to which IgE antibody is only rarely found in peanut allergic patients. [Cabanos C, Protein Expr Purif 2010]

Peanut Component Analysis Ara h2 identified as most important component for peanut allergy in several studies (Nicolaou et al, 2010, and Asarnoj et al, 2010) Guidance for use in clinical practice is lacking Peanut Components (Individual allergenic proteins) Family Ara h 1, 2, 3, 4, 6 and 7 Seed storage protein Ara h9 Non-specific lipid transfer protein Ara h8 Pathogenesis related 10 family (highly cross-reactive with Bet v 1)

Niacolaou N et al. JACI 125:191-197, 2010 Differentiation of Peanut Allergy from Tolerance using Component Resolved Diagnostics Children recruited prenatally; evaluated at 1, 3, 5, 8 yrs Peanut sensitization identified by PST (>3mm) or ImmunoCAP (>0.2 kUa/L); n=108 1. Peanut allergic: Hx of reaction and IgE anti-peanut > 15 kUa/L or PST >8 mm; or failed peanut challenge (n=29) 2. Peanut tolerant: sensitized & negative peanut challenge (n=52) 10% of 8yr children were sensitized; ~2% peanut allergic Microarray component resolved IgE identified Arah2 as the single best discriminator of tolerance vs allergy and the most important predictor of clinical allergy to peanut Niacolaou N et al. JACI 125:191-197, 2010

Component Resolved Diagnostics Utility Nicolaou et al Component Resolved Diagnostics Utility Nicolaou et al. JACI 125:191-7,2010 Child, + peanut allergy history, + PST to peanut Prognosis (risk for systemic reaction) can be very different if sensitized to Ara h 8 [Bet v 1 (PR10) like protein] {minimal systemic reaction risk {marker for primary sensitization to birch/alder pollen} Ara h 1, 2, 3 [seed storage proteins] high risk {Ara h 2 is considered a risk marker for severe allergic reactions} Ara h 9 [lipid transfer protein] high risk {suggests primary sensitization to peach or other LPT-containing fruits}

Ara h 9 + (LTP) Ara h 1,2,3 +/- Ara h 8 – (PR10) Primary sensitization Risk of severe reactions Ara h 9 - Ara h 1,2,3 + Ara h 8 - + Peanut Extract Specific IgE Ara h 9 - Ara h 1,2,3 - Ara h 8 + Secondary sensitization Cross-reactivity with birch pollen Local reactions (OAS)

Clinical Decision Points Confirming Allergy Ara h2 ≥ 2 kU/L PPV 89% 50% of failed challenges avoided Excluding Allergy Ara h2 ≤ 0.1 kU/L NPV 91% Ara h1, Ara h2, and Ara h3 all ≤ 0.1 kU/L NPV 93%

Hazelnut

JACI-Practice 2:633-4, 2014 Cor a 1 = Bet v 1-PR10-Family Cor a 8 = Lipid Transfer Protein Cor a 9 = 11S globulin >2 kUa/L -> Cor a 14 = 2S albumin >1 kUa/L -> (Dx Sensitivity: 92%, Dx Specificity 93% for clinical reactivity to hazelnut 116 patients + Hx of hazelnut allergy Oral food challenge with hazelnut ~9% of US children outgrow tree nut 42=>IgE anti-HN extract & components allergy/ limitations of SPT/IgE

Cor a 8+ (LTP) Cor a 9/14 +/- Cor a 1- (PR10) Primary sensitization Risk of severe reactions Cor a 8 - Cor a 9/14 + Cor a 1 - + Hazelnut Extract Specific IgE Cor a 8 - Cor a 9/14 - Cor a 1 + Secondary sensitization Cross-reactivity with birch pollen Local reactions (OAS)

Diagnosis of Human Allergic Disease Multiplex Chip Technology Using Allergen Components ImmunoCAP ISAC ® Characterisation of IgE First allergens cloned Diagnostic recombinant allergen panels Provocation testing RAST* First allergen chip 1880 1967 1988-91 1995-1999 2000 2007 In-vivo testing In-vitro testing Component-resolved diagnosis

ThermoFisher Scientific ISAC® Technology Incubate sample Stain with SAb Prepare serum Scan biochip Analyse images Duration: 3 hours Duration: 10 min. Create report Wash 62

Improved IgE Antibody Crossreactivity Assessment ISAC Multiplex microarray using component allergens PR10 Family (Bet-v1, Cor-a1; Mal-d1; Pru-p1; Gly-m4; Ara-h8; Apr-g1; Dau-c1, Act-d8) Profilins (Bet-v2; Ole-e2; Hev-b8; Phl-p12) Lipid Transfer Proteins (Cor-a9;Pru-p3;Art-v3;Par-j2) Calcium binding Proteins (Bet-v4; Phl-p7) Serum Albumin Family (Bos-d6; Fel-d2; Can-f3; Equ-c3; Gal-d5) Tropomyosins (Pen-a1;Der-p10;Bla-g7;Ani-s3)

Molecular Allergen Specific IgE Analyses ImmunoCAP-ThermoFisher Scientific/ Phadia Singleplex IgE Antibody Assay Immulite-Siemens Singleplex IgE Antibody Assay Immulite Siemens ISAC Multiplex IgE Antibody Assay Basic reagents and chemistries are similar * Allergen on solid phase binds antibody * Buffer wash to remove unbound protein * Enzyme anti-IgE detects bound IgE * Response interpolated from a reference curve Units, degree of quantitation, analytical sensitivity differ: 0.1 kUa/L vs 0.3 ISU *

Strengths and Weaknesses of Allergen Component-based ISAC Analysis 1. Identifies IgE anti-food/pollen allergen cross-reactivity 2. Can detect relevant IgG and IgA blocking antibodies Weaknesses: 1. Analytical sensitivity lower for some allergens than extract based autoanalyzers 2. Will not detect IgE antibodies of all specificities in a given allergen extract unless all allergens are on the chip 3. IgE binding can be interfered by antibodies of same specificity but different isotypes (e.g. post immunotherapy)

missing or low abundance Rationale for Use of Molecules Allergen source/extract (A, B, C) A a1 B b1 C c1 a3 c2 a2 b2 b3 Reasoning for using allergen molecules missing or low abundance defined clinical risk/role IgE cross- reactivity genuine (primary) sensitization Allergen molecules b3 c1 a2 a3 c2 The consequence of adding missing or low abundance allergens is inproved limit of quantitation. By detecting <limit of quantitation >analytical specificity <limit of quantitation >analytical specificity Effects on assay results marker of cross-reactivity primary allergen Bet v 1 homologues Ara h 8-peanut Pru p 1-peach Cor a 9,14 Hazelnut Examples: Kleine-Tebbe et al. 2015 Gly m 4-soy Cor a 1-hazelnut Ara h 1,2,3,6 Peanut

Rationale for Use of Extracts versus Molecules A. Sensitization profile = IgE repertoire B. Associated risks for clinical reactions extracts Monosensitization low utility utility Oligosensitization moderate Polysensitization high molecules C. Abundance in whole extract D. Stability of single allergen extracts high high utility utility moderate moderate low low molecules Kleine-Tebbe et al. 2015

Singleplex Assays with quantification in Trends in IgE Antibody Serology Extract Based Singleplex Assays with quantification in kUa/L IgE Antibody Assays Multiplex Chip-Based ISAC Allergenic Molecules (components)

Summary: Molecular Allergology Optimize analytical sensitivity by replacement or supplementation of relevant allergens in test extracts Standardize allergen extracts More precisely defining genuine sensitization for complex patients sensitized to many allergen groups Identify of cross-reactions and predictive risk factors Since no definitive single criterion proves the existence of allergic disease, IgE antibody results should be reported as analogue data, and viewed only as one risk factor for allergic disease

Theoretical logistic relationship between IgE antibody concentration and presence (risk) of allergic disease Dynamic changes in IgE antibody affinity, specificity, and specific activity continually alter this relationship Dynamic changes in a patient’s age and type of allergic disease continually alter this relationship

Probability Risk Based Assessment of Allergic Disease Serum IgE anti-egg, milk, peanut, fish , soy, wheat allow improved probably assessment of children for clinical sensitivity to foods. (Sampson and Ho. JACI: 1997:100:444-51; Sampson JACI: 2001:107:891-6)

Probability-based Risk Assessment: Quantitative Allergen-specific IgE Results 95% Predictive Values: Egg: 7 kIU/L, milk: 15 kIU/L, Peanut 14 kIU/L, Fish 10 kIU/L, Wheat: 80 kIU/L, Soy 65 kIU/L (Sampson: 2001)

decision point %/Specific IgE (kU/L) Published Milk-Specific IgE Antibody Predictive Values for Positive Cow’s Milk Provocation Test Study Age- years No. subjects Study design Oral Milk Challenge Positive Predictive decision point %/Specific IgE (kU/L) Sens. for IgE level Spec. for IgE level Sampson and Ho 1 5.2 avg 196 Retrospective DBPCFC 95% : 32 90% : 23 51% 58% 98% 94% Sampson 2 3.8 med 100 Prospective 95% : 15 57% Garcia-Ara C et al.3 0.4 avg. 170 Open controlled 95% : 5 90% : 2.5 30% 48% 99% 95% Garcia-Ara et al.4 0.4 avg - start 66 follow-up 95% : 2.7 for age 1.1-1.5 95% : 9 for age 1.6-2 95% : 24 for age 2.1-3 76% 59% 60% 3% 90% 100% Celik-Bilgili S et al.5 1.1 med 501 or open 90% : 88.8 * van der Gugten et al.6 3.0 avg 213 95% : 52 90% : 26.8 95% : >100 90% : 66.4

Singleplex Assays with quantification in Trends in IgE Antibody Serology Extract Based Singleplex Assays with quantification in kUa/L IgE Antibody Assays Multiplex Chip-Based ISAC Allergenic Molecules (components)