Food Allergens : Methods and Protocols 2717

This detailed volume provides a comprehensive collection of methods and protocols in food allergy and food allergens studies. The selected protocols explore the study of food allergens, from recombinant production, purification procedures, IgE and T cell epitopes characterization, to allergen structure description, cellular responses, and tolerance induction, through a variety of techniques and animal models. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Food Allergens: Methods and Protocols serves as an ideal reference for scientists at all stages involved in the study of food allergy and allergenic components. Preface Contents Contributors Chapter 1: Food Allergens of Plant and Animal Origin: Classification, Characteristics, and Properties 1 Food Allergy and Its Prevalence 2 The Big Eight in Food Allergy: Peanut, Tree Nuts, Milk, Egg, Fish, Shellfish, Soybean, and Wheat 3 Common and Differential Properties of Food Allergens 4 Food Allergens Classification 4.1 Classification of Food Allergens of Plant Origin 4.1.1 Superfamily of Cupins 4.1.2 Superfamily of Prolamins 4.1.3 Pathogenesis-Related Proteins 4.1.4 Additional Families 4.2 Classification of Food Allergens of Animal Origin 5 Conclusions References Chapter 2: Purification of Food Allergens from Their Natural Sources: Chromatographic Methods 1 Introduction 2 Materials 2.1 Preparation of Flours 2.2 Protein Solubilization and Extraction 2.3 Protein Purification 3 Methods 3.1 Flour Preparation (Room Temperature) 3.2 Column Packing 3.3 Target Protein Solubilization and Purification (See Note 11) 3.4 Protein Purity Assessment 4 Notes References Chapter 3: Recombinant Production of Food Allergens in Yeast Pichia pastoris 1 Introduction 2 Materials 2.1 P. pastoris Strain and Plasmid 2.2 Nucleotide Sequences of Oligonucleotide Primers 2.3 PCR Screening of Yeast Colonies and PCR Reaction Mix 2.4 Electroporation 2.5 Yeast Culture Media 2.5.1 Reagents for Culture and Selective Media 2.5.2 Reagents and Media for Expression of Recombinant Protein 2.6 Other Reagents 3 Methods 3.1 Preparation of Competent Pichia pastoris Cells (KM71) (See Note 8) 3.2 Preparation of Plasmid DNA for Transformation 3.3 Transformation of KM71 by Electroporation 3.4 Preparation of A Master Plate for PCR Screening and Protein Expression 3.5 Screening of Yeast Colonies by PCR 3.6 Small-Scale Expression of Recombinant Allergen in Yeast P. pastoris Cells 4 Notes References Chapter 4: Yeast Surface Display Methodology for the Characterization of Food Allergens In Situ 1 Introduction 2 Materials 2.1 Strains 2.2 Patient Sera Collection 2.3 Antisera Production 2.4 Cloning of Allergen 2.5 Transformation of Yeast Cells EBY 100 and Allergen Expression 2.6 Flow Cytometry Analysis of Allergen Yeast Surface Display 2.7 Solubilization of Act d 1 2.8 Allergen Proteolytic Activity Detection 2.9 Dot Blot and Immunoblot Analysis 2.10 Direct ELISA and ELISA Inhibition 3 Methods 3.1 Collection of Patient ́s Sera 3.2 Antisera Production 3.3 Cloning of Allergen 3.4 Transformation of Yeast Cells EBY 100 and Allergen Expression 3.5 Flow Cytometry Analysis of Allergen Yeast Surface Display 3.6 Solubilization of rAct d 1 3.7 Proteolytic Activity Detection of Allergen 3.8 Dot Blot and Immunoblot Analysis 3.9 Direct ELISA and ELISA Inhibition 4 Notes References Chapter 5: Characterization of Linear IgE-Binding Epitopes in Food Allergens 1 Introduction 2 Materials 2.1 Peptide Synthesis 2.2 Dot Blot 3 Methods 3.1 Generation of the Peptide List 3.2 SPOT Synthesis of Peptides (See Note 10) 3.2.1 Derivatization of the Cellulose Membrane 3.2.2 Generation of the SPOTs Array 3.2.3 Assembly of Peptides on SPOTs 3.2.4 Final Fmoc and Side-Chain Deprotection 3.3 Dot Blot 3.4 Mutational Analysis 4 Notes References Chapter 6: Characterization of T-Cell Epitopes in Food Allergens by Bioinformatic Tools 1 Introduction 2 Materials 2.1 UniProt 2.2 Immune Epitope Database (IEDB) 2.3 Research Collaboratory for Structural Bioinformatics (RCSB), Protein Data Bank (PDB) 2.4 IL4pred 2.5 AllerTop v.2.0 2.6 Discovery Studio Software 3 Methods 3.1 Protein Sequence Retrieval 3.2 T-Cell Epitope Prediction by ``MHC-II Binding Predictions ́ ́ Tool 3.3 Evaluation of the Ability to Induce IL-4 3.4 Allergenicity Evaluation 3.5 Epitope Population Coverage Analysis 3.6 HLA-Epitope Molecular Docking 4 Notes References Chapter 7: Phage Immunoprecipitation Sequencing (PhIP-Seq) for Analyzing Antibody Epitope Repertoires Against Food Antigens 1 Introduction 1.1 Autoantigen Discovery 1.2 Viral Antigen Discovery 1.3 Antibacterial Antibody Responses 1.4 Food Allergens 2 Experimental and Data Analysis Aspects in PhIP-Seq Experiments 2.1 Key Steps of the Experimental PhIP-Seq Workflow 2.1.1 Library Design 2.1.2 Phages-Antibody Binding and Immunoprecipitation 2.1.3 PCR Amplifications of Libraries 2.2 Data Analysis 2.2.1 Generalized Poisson Model and True Hit Calculation 2.2.2 Z-Score-Based Model 2.2.3 Novel Methods (Gamma Poisson, BEER, AVARDA) 2.2.4 Machine Learning Approaches with PhIP-Seq Data 3 Advantages and Limitations of PhIP-Seq 4 Outlook References Chapter 8: 1D-, 2D-Gel Electrophoresis, Immunoblotting, and Enzyme-Linked Immunosorbent Assay (ELISA) for the Study of Food Al... 1 Introduction 1.1 Protein Extraction 1.2 1D/2D-Electrophoresis 1.3 Immunoblotting 1.4 ELISA 2 Materials 2.1 Protein Extraction 2.2 1D-Electrophoresis 2.3 2D-Electrophoresis 2.4 Immunoblotting 2.5 ELISA 3 Methods 3.1 Protein Extraction 3.2 1D-Electrophoresis 3.3 2D-Electrophoresis 3.4 Immunoblotting 3.5 ELISA 4 Notes References Chapter 9: Preparation of Blinded Food Matrixes for Clinical Oral Challenges 1 Introduction 2 Materials 2.1 Food 2.2 Matrix 2.3 Blinding 3 Methods 3.1 Doses 3.2 Manufacturing 3.3 Example #1-Preparation of Oral Soy Flour Challenge in Granola Matrix 3.4 Example #2-Preparation of Shrimp Burgers 4 Notes References Chapter 10: Structural Characterization of Food Allergens by Nuclear Magnetic Resonance Spectroscopy 1 Introduction 2 Materials 2.1 Isotope-Labeled Expression 2.2 Protein Purification 2.3 NMR Experiments 2.3.1 NMR Materials 2.3.2 NMR Software 2.3.3 Software for Structure Calculation 2.3.4 Backbone Relaxation Dispersion Experiments 2.3.5 Ligand Titration 2.3.6 Ligand-Detected Relaxation Dispersion Experiments 3 Methods 3.1 Isotope-Labeled Expression 3.2 Protein Purification 3.3 NMR Measurements 3.3.1 NMR Experiments 3.3.2 Structure Determination 3.3.3 Backbone Relaxation Dispersion Experiments 3.3.4 Ligand Titration 3.3.5 Ligand-Detected 1H Relaxation Dispersion Experiments 4 Notes References Chapter 11: Coculture of Human Dendritic and T Cells for the Study of Specific T Cell-Mediated Responses Against Food Allergens 1 Introduction 2 Materials 3 Methods 3.1 PBMC Isolation from Human Blood 3.2 Isolation and Generation of Human DCs 3.2.1 Direct Isolation of DCs from PBMCs 3.2.2 In Vitro Generation of Mo-DCs 3.2.3 In Vitro Generation of DCs from Human Monocytic (THP-1) Cell Line 3.3 Direct Isolation of CD4+ T from PBMCs 3.4 Determination of DC and CD4+ T Cell Purity by Flow Cytometry 3.5 Evaluation of the DC Uptake Capacity 3.6 Coculture of DCs and CD4+ T Cells for the Evaluation of Allergen Responsiveness 3.7 Approaches for the Evaluation of the Allergen-Specific CD4+ T Cell Responses 3.8 Early T Cell Responses (6-18 h) 3.8.1 Upregulation of Activation Markers 3.8.2 Intracellular Cytokine Staining 3.8.3 Cytokine Quantification by ELISpot 3.9 Delay T Cell Responses (24 h-5 Days) 3.10 Benefits and Limitations of DC:T Cell Cocultures 4 Notes References Chapter 12: Evaluation of the Suppressive Capacity of Regulatory T Cells in Food Allergy Research 1 Introduction 2 Materials 2.1 Materials for Experiments with Mice 2.2 Materials for Experiments with Human Samples 2.3 Determination of Treg and Tconv Cell Purity 3 Methods 3.1 T Cell Isolation from Mouse Spleen 3.1.1 Mice Handling (See Note 6) 3.1.2 Splenocytes Isolation 3.1.3 Purification of Murine Treg/Tconv Cells 3.2 T Cell Isolation from Human Peripheral Blood 3.2.1 Peripheral Blood Mononuclear Cells (PBMCs) Isolation from Human Whole Blood 3.2.2 Purification of Human Treg/Tconv Cells 3.3 Determination of Treg and Tconv Cell Purity 3.4 CFSE Staining Protocol for Murine and Human Tconv 3.5 Immunosuppression Assay for Murine and Human Tregs 3.6 Flow Cytometry Analyses of the Immunosuppression Assay for Murine and Human Tregs 3.7 Limitations of the Described Immunosuppression Assay for Mouse and Human Tregs 4 Notes References Chapter 13: Mass Cytometry in Food Allergy Research 1 Introduction 2 Mass Cytometry in Food Allergy Research 2.1 Mass Cytometry in Non-IgE-mediated Food Allergy 2.2 Changes in Immune Profiles Upon Immunotherapy 2.3 Insights of Mass Cytometry on Exposures and Immune Function 3 Technical Notes References Chapter 14: Indirect Basophil Activation Test for Peanut Allergy Diagnosis Using Human Donor Basophils 1 Introduction 2 Materials 2.1 Collection of Donor Basophils 2.2 Stripping and Resensitization of Donor Basophils 2.3 Basophil Activation Test 3 Methods (See Note 1) 3.1 Basophil Isolation 3.2 Stripping and Resensitization of Donor Basophils 3.3 Basophil Activation Test 4 Notes References Chapter 15: Standardization of Food Allergen Measurements Using Multiplex Array Technology 1 Introduction 2 Materials 2.1 Reagents Supplied for MARIA for Foods 2.2 Instrumentation and Reagents Not Supplied 3 Methods 3.1 Sample Extraction (See Note 1) 3.2 MARIA for Foods Assay 4 Notes References Chapter 16: Biosensors for the Detection of Food Allergens 1 Introduction 2 Materials 3 Methods 3.1 Fabrication of the Aptasensor 3.1.1 Cleaning of SPR Au Interfaces 3.1.2 Functionalization with Thiol 3.1.3 Functionalization with Neutravidin 3.1.4 Aptamer Immobilization 3.2 Wine Pretreatment 3.3 SPR Measurements 3.4 Calibration of the Aptasensor 3.5 Analysis of Lysozyme in Wine 3.6 Analysis of Lysozyme in Food 3.7 Studying the Interaction of Lysozyme with Phenolic Compounds in Wine 4 Notes References Chapter 17: Standardization of a Mass Spectrometry-Based Workflow for Food Allergen Quantification 1 Introduction 2 Materials 2.1 Composition of Extraction Buffer and Protein Extraction Conditions 2.2 Disposable Tools for Partial Protein Purification 2.3 Controlled Enzymatic Digestion and Digest Clean-up 2.4 Bioinformatic Tools 3 Methods 3.1 Peptide Marker Selection 3.1.1 In Silico Prediction-Based Approach 3.1.2 Literature Review-Based Approach 3.1.3 Evidence-Based Approach 3.2 MS Setup and Chromatographic Separation 3.3 Optimization of the Sample Preparation Protocol 3.3.1 Sample Preparation and Homogeneity Assessment 3.3.2 Extraction/Purification Conditions 3.3.3 Specific Enzymatic Digestion Conditions 3.3.4 Purification at Peptide Level 3.3.5 Refining List of Markers 3.4 Method Validation 4 Notes References Chapter 18: Identifying Similar Allergens and Potentially Cross-Reacting Areas Using Structural Database of Allergenic Protein... 1 Introduction 2 Materials 3 Methods 3.1 Identifying Allergens Similar to a Given Protein Using the FASTA Search in SDAP 3.2 Finding a 3D-Structure for Your Protein in SDAP 3.3 Matching Potential IgE Epitopes to Your Protein 3.4 Finding Sequences in Other Allergens Similar to Known Epitopes: The Peptide Similarity Search 3.5 Determining the PD Between Two Different Peptides or One Peptide to Another Protein 3.6 Making a PD-Graph of Similar Peptides with D-Graph 3.7 Conclusions References Chapter 19: Validation Procedures for Quantification of Food Allergens by Enzyme-Linked Immunosorbent Assay (ELISA) 1 Introduction 2 Materials 2.1 Sample Preparation 2.2 Antigen Extraction 2.2.1 Protein Extraction Buffers 2.2.2 Other Materials and Equipment 2.3 ELISA Operation 3 Methods 3.1 Sample Preparation 3.1.1 Preparation of Processed Samples 3.1.2 Preparation of Spiked Samples 3.1.3 Preparation of Incurred Samples 3.1.4 Preparation of Sample Flours 3.1.5 Preparation of Protein Extracts 3.2 ELISA Procedures 3.3 ELISA Validation Procedures 3.3.1 Antibody and Standard Information 3.3.2 Calibration Curve and Quantification Range 3.3.3 Limit of Detection (LOD) and Limit of Quantification (LOQ) 3.3.4 Specificity and Cross-Reactivity 3.3.5 Reproducibility 3.3.6 Recovery 3.3.7 Assay Applicability and Robustness 3.3.8 Ruggedness, Shelf-Stability, and Lot-to-Lot Variability 4 Notes References Chapter 20: Detection of Bet v 1 Homologous Proteins and Plant Profilins by Indirect ELISA 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 21: A Mouse Model of Shrimp Allergy with Cross-Reactivity to Crab and Lobster 1 Introduction 2 Materials 2.1 Allergen Extracts 2.2 SDS-PAGE Gels 2.3 Mouse Sensitization 2.4 Shrimp-, Lobster-, and Crab-Specific IgE ELISAs 2.5 Mouse Challenges 3 Methods 3.1 Resuspend Shrimp, Lobster, and Crab Extracts, and Identify Tropomyosin 3.2 Sensitize Mice to Shrimp 3.3 Shrimp-, Lobster-, and Crab-Specific IgE ELISAs 3.4 Shrimp Challenge 4 Notes References Chapter 22: Animal Models in the Study of Food Allergens: Long-Term Maintenance of Allergic Reactivity in Mouse Models of Food... 1 Introduction 2 Materials 2.1 Preparation of Peanut Extract (PE) from Peanut Flour (See Note 1) 2.2 Sensitizing Mice to Ovalbumin 2.3 Sensitizing Mice to Peanut 2.4 Enzyme-Linked Immunosorbent Assay (ELISA) 2.5 Oral Food Challenge 2.6 Analysis of Mast Cell Degranulation 2.7 Monitoring of Anaphylaxis 3 Methods 3.1 Preparation of Peanut Extract (PE) from Peanut Flour (See Note 1) 3.2 Systemic Sensitization with OVA and Alum 3.3 Oral Sensitization with Peanut Extract and Cholera Toxin 3.4 Confirmation of Sensitization by ELISA Analysis of Allergen-Specific IgE (See Note 11) 3.5 Oral Food Challenge (OFC) and Monitoring of Anaphylaxis 4 Notes References Chapter 23: Study of MicroRNAs Expression in Food Allergy 1 Introduction 2 Materials 2.1 Next-Generation Sequencing 2.2 RNA Extraction 2.3 Reverse Transcription 2.4 Quantification of miRNAs by qPCR 3 Methods 3.1 Determination of the miRNA Profile by NGS 3.2 miRNA Extraction 3.2.1 Serum miRNAs Extraction 3.2.2 Intracellular miRNAs Extraction 3.3 Reverse Transcription 3.4 Quantification of miRNA Levels by qPCR 4 Notes References Chapter 24: De Novo Transcriptomic Analyses to Identify and Compare Allergens in Foods 1 Introduction 2 Materials 3 Methods 3.1 RNA-Seq Data Acquisition 3.2 RNA-Seq Correction 3.3 De Novo Transcriptome Assembly 3.4 De Novo Transcriptome Assembly Quality Analysis 3.5 Transcriptome Completeness Analysis 3.6 Allergen Reference Database Construction 3.7 BLAST Search for Potential Allergens 3.8 Processing/Filtering BLAST Search Results 3.9 Grouping Allergens into Known Allergens, Highly Likely Allergens, and Likely Allergens 3.10 Categorizing New Allergens Based on the Origin Organism 3.11 Comparing Putative New Allergens to Homologous Allergens in Other Food Sources 3.12 Assessing the Allergenicity Potential of Contig from Transcriptome Using AllerCatPro 2.0 4 Notes References Chapter 25: Chromatin Immunoprecipitation Sequencing (ChIP-Seq) Assay in Food Allergy Research 1 Introduction 2 Materials 3 Methods 3.1 Day 1: Chromatin Cross-Linking, Chromatin Shearing, and Antibody Binding 3.2 Day 2: Immunoselection and Chromatin De-Cross-Linking 3.3 Day 3: DNA Purification and Library Preparation and Cleanup 3.4 Bioinformatic Analysis 4 Notes References Index