Development of multi-analyte tools for food allergen analysis

Transcription

1 Development of multi-analyte tools for food allergen analysis James Hindley, PhD Executive Director, Indoor Biotechnologies Ltd

2 Objectives Module 4 Analysis of Allergens in Food Multi-analyte allergen analysis Clinically-relevant analysis 1. To compare best practices for measuring food allergens. 2. To develop assays for specific food allergens e.g. Ara h 2, Bos d 5 3. To compare specific peanut allergen levels in: Foods containing peanut (butter, flour, oils) Allergenic extracts Preparations used for oral immunotherapy or allergy prevention 4. To develop multiplex technology for measuring food allergens.

3 Immunoassays for Allergen Testing Food Industry Generic ELISA (peanuts, milk, egg..) Measures total protein in ppm Pharma/Diagnostics Industry Specific ELISA defined targets (Ara h 2, Ara h 6) Measures specific allergen ng and µg of Ara h 2 Polyclonal several targets Monoclonal single target Standard = total food extract Standard = purified allergen

4 Aim To develop accurate, sensitive and reliable immunoassays for the simultaneous quantification of clinically relevant food allergens.

5 Our approach to multiplex assay development Purified Allergen Monoclonal Antibodies ELISA Multiplex Assay Food Allergen Peanut Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 8, Ara h 9 Shellfish Tropomyosin Milk Bos d 4, Bos d 5, Bos d 6, Bos d 8 Egg Gal d 1, Gal d 2, Gal d 4 Hazelnut Cor a 1, Cor a 8, Cor a 9, Cor a 14 Cashew Ana o 3 Soy Gly m 4, Gly m 5, Gly m 6 Wheat Tri a 14, Gliadin, Tri a 19 Fish Gad c 1 ~90% of all food allergies in the US Most clinically relevant proteins from those foods

6 Purified food allergen proteins - active ingredients Description Source Description Source MILK ALLERGENS Natural Bos d 4 Cow's milk α-lactalbumin HAZELNUT ALLERGENS Recombinant Cor a 1 Pichia pastoris; PR-10 Natural Bos d 5 Cow's milk β-lactoglobulin Recombinant Cor a 8 Pichia pastoris; nsltp Natural Bos d 6 Cow's milk serum albumin Natural Cor a 9 Hazelnut flour 11S globulin Natural Bos d 8 Cow's milk casein Recombinant Cor a 14 Pichia pastoris; 2S albumin EGG ALLERGENS LoTox ngal d 1 LoTox ngal d 2 LoTox ngal d 4 FISH ALLERGENS Recombinant Gad c 1 Chicken egg ovomucoid Chicken egg ovalbumin Chicken egg lysozyme Pichia pastoris; parvalbumin CRUSTACEAN SHELLFISH ALLERGENS Natural tropomyosin Shrimp (white and/or brown) PEACH ALLERGENS Recombinant Pru p 3 CASHEW ALLERGENS Recombinant Ana o 3 Pichia pastoris; nsltp Pichia pastoris; 2S albumin PEANUT ALLERGENS Natural Ara h 1 Natural Ara h 2 Natural Ara h 3 Natural Ara h 6 Recombinant Ara h 2 Recombinant Ara h 8 Recombinant Ara h 9 LoTox Peanut Antigen WHEAT ALLERGENS Recombinant Tri a 19* SOYBEAN ALLERGENS Recombinant Gly m 4 Natural Gly m 5 Natural Gly m 6* Blanched peanut cupin (7S globulin) Peanut flour, light roast conglutin (2S albumin) Blanched peanut cupin (11S globulin) Peanut flour, light roast conglutin (2S albumin) Pichia pastoris; conglutin (2S albumin) Pichia pastoris; PR-10 Pichia pastoris; nsltp Peanut flour, light roast Pichia pastoris; omega-5 gliadin Pichia pastoris; Soybean PR-10 Soybean flour β-conglycinin (7S globulin) Soybean flour Glycinin (11S globulin) WHO/IUIS Allergen Nomenclature Database:

7 Purity of Food Allergen Proteins Assessed by Mass Spec Allergen Sequence coverage Relative Abundance nbos d 4 63% 97% nbos d 5 87% 99% nbos d 8 64% 99% ngal d 2 87% 99% ngal d 4 92% 92% Shrimp Tropomyosin 93% 99% rpru p 3 99% 99% rana o 3 87% 97% Allergen Sequence coverage Relative Abundance rcor a 1 100% 99% rcor a 8 95% 99% rcor a 14 71% 99% nara h 1 73% 84% nara h 2 86% 96% nara h 3 85% 94% rara h 8 81% 96% rara h 9 100% 99% rgly m 4 77% 98% rgly m 5 56% 92%

8 Purified Food Allergens and ELISA Affinity and/or HPLC purified Purity checked by SDS-PAGE and Mass Spec Validated by IgE binding ISO 9001:2008 Certified LoTox TM Low Endotoxin Allergen specific ELISA 2.0 Pre-coated plates includes all buffers & reagents Fully validated

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10 Peanut Flours Characterisation of Peanut Products Filep et al, JACI 2017 Diagnostic Extracts Immunotherapies Oral

11 Learning Early About Peanut Allergy: (LEAP - trial of prevention of peanut allergy)

12 Characterisation of Bamba Hindley et al, JACI 2017 Bamba US/UK samples ErdnußLocken Germany Bamba is a well formulated peanut product, with each of the three major allergens present in uniform amounts. Cumulative weekly doses of ~330 mg peanut allergens are associated with prevention of peanut allergy. Estimated Doses of Peanut Allergens in Bamba Administered During LEAP Dose Ara h 1 (mg) Ara h 2 (mg) Ara h 6 (mg) Mean allergen/stick* sticks** One serving sticks*** Median weekly dose sticks x 52 weeks*** 12 months consumption 4,326 6,224 6,604

13 Analysis of Peanut Allergens in House Dust Dust samples from MAAS (n = 239) analyzed by ELISA R = 0.70; p<0.001 Samples provided by Drs. Adnan Custovic and Angela Simpson

14 Analysis Of Allergens In Baked Milk Muffins Baked milk materials such as muffins often used for oral food challenges Gold standard in US Baked milk muffin safe Recent death of 3 year old boy in US following routine challenge* * Roberts G. Food challenges, the risks and benefits. Clin Exp Allergy. 2017;47:1106.

15 Analysis Of Allergens In Baked Milk Muffins Measurement of major milk allergens using ELISA Allergen name Protein type % of milk allergic sensitised* Bos d 5 Beta Lactoglobulin 62% Bos d 11 Beta Casein 57% *EAACI Molecular Allergology User s Guide. Bos d 5 Bos d µ g/g 400 µ g/g Uncooked Baked 0 Uncooked Baked

16 Analysis Of Allergens In Baked Milk Muffins Bos d 5 Bos d µ g/g 400 µ g/g Uncooked Baked Top Baked Middle Baked Bottom Uncooked Baked Top Baked Middle Baked Bottom

17 Analysis Of Allergens In Baked Milk Muffins 4000 Bos d Max = 3389µ g/g µ g/g x 2.4 Min = 1391µ g/g 4000 Bos d 11 P value < Batch A Batch B Batch C Batch D 3000 µ g/g M t S in ai NHS

18 Analysis Of Allergens In Baked Milk Muffins Average amount of allergen Total weight of baked muffin = 62 grams Bos d 5 Bos d 11 Amount of allergen per gram of baked muffin 0.07 µg/g 2,841 µg/g Amount of allergen per baked muffin 4 µg 176,142 µg

19 Food Multiplex Assay Development Purified natural allergens as standards Purified by affinity chromatography/hplc Validated for IgE binding Purity checked by SDS-PAGE and mass spec Quantified by AA analysis Purified allergen specific antibody pairs Preferably monoclonal Ara h 6 Bos d 5 Gal d 2 STM Food Target protein Standard Capture Antibody Detection Antibody Peanut Ara h 6 ST-AH6 mab 3B8 mab 3E12 Milk Bos d 5 ST-NBD5 mab 97N mab 117N Egg Gal d 2 LTN-GD2-1 HYB HYB Shrimp Tropomyosin ST-STM mab 1A6 pab

20 MARIA standard curves 120 Standard curves range between: Peanut, Ara h 6 Milk, Bos d 5 Egg, Gal d 2 Shrimp, STM Mean Fluorescence Intensity (normalised) Allergen Concentration (ng/ml)

21 Performance Characteristics Lower Limits of Quantification between 20ng/ml and 20pg/ml Multiplex Assay is up to 40 fold more sensitive than ELISA Good reproducibility (Average CVs <14%) Lower Limit of Quantification (LLOQ) Reproducibility (%CV) Food Type Target Protein (allergen) ng/ml mg/kg Intra-assay Inter-assay Parallelism Peanut Ara h Milk Bos d Egg Gal d Shrimp STM

22 Characterisation of ifaam materials Concentrations (µg/g) of specific food allergens Food Type: Peanut Milk Egg Shrim p Target Protein (Allergen): Ara h 6 Bos d 5 Gal d 2 STM ifaam Cookie < 2.3 ifaam Chocolate Dessert < 2.3 ifaam Chocolate Bar < 2.3 Shrimp Extract <0.02 < 0.1 < ,42 0

23 Validation with ifaam food matrices Choc Bar Ara h 3 (µ g /g) Peanut Peanut Milk Ara h 3 Ara h 6 Bos d 5 Ara h 3 Chocolate Bar Ara h 6 (µ g/g) Ara h 6 Chocolate Bar Bos d 5 (µ g/g) Bos d 5 Chocolate Bar Choc Dessert PPM Ara h 3 Dessert PPM Ara h 6 Dessert PPM Bos d 5 Dessert Ara h 3 (µ g/g) Ara h 6 (µ g/g) 3 2 Bos d 5 (µ g/g) PPM PPM PPM

24 Immunoassays for food Immunoassay Formats Food Source Allergen ELISA 2.0 MARIA Peanut Ara h 1 In progress Ara h 2 In progress Ara h 3 Ara h 6 Ara h 8 In progress Shellfish Tropomyosin Milk Native Bos d 5 Bos d 8 (Casein) In progress In progress* Hazelnut Cor a 9 Cashew Ana o 3 Egg Gal d 2 In progress In progress* Soy Gly m 4 In progress In progress Gly m 5 In progress In progress Wheat Tri a 19 Planned Planned Fish Gad c 1 (Parvalbumin) Planned Planned *By 2018

25 Conclusions An extensive panel of purified food allergens and allergen specific immunoassays (ELISA and MARIA) have been developed sensitive, quantitative, reproducible and validated Peanut foods, allergenic extracts and OIT formulations show extensive variability in allergen content and ratios of major allergens. These tools will: Aid selection, standardization and formulation of food allergy diagnostics and OIT products Allow risk of environmental exposures to be linked to allergic sensitization Improve risk assessment of allergic reactions due to allergen contaminants in the food industry.

26 Acknowledgments Indoor Biotechnologies Ltd. UK Anna Kuklinska-Pijanka Max Bermingham Maria Oliver Ross Yarham Izabel Alvares Angharad Cullinane Hayley Gallagher-Mills Indoor Biotechnologies Inc. USA Martin Chapman Bryan Smith Stephanie Filep Anna Pomes Sabina Wuenschmann Eva King Kristina Reid Black Denise Block University of North Carolina Scott Commins Brian Vickery Michael Kulis Guy s & St. Thomas NHS Trust Mary Feeney George Du Toit Gideon Lack University of Manchester, UK Adnan Custovic Angela Simpson NIH-National Institute of Allergy and Infectious Diseases NIH Award R01AI