Correlation of IgE/IgG4 milk epitopes and affinity of milk-specific IgE antibodies with different phenotypes of clinical milk allergy Julie Wang, MD,

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Correlation of IgE/IgG4 milk epitopes and affinity of milk-specific IgE antibodies with different phenotypes of clinical milk allergy Julie Wang, MD, Jing Lin, PhD, Ludmilla Bardina, MSc, Marina Goldis, MD, Anna Nowak-Węgrzyn, MD, Wayne G. Shreffler, MD, PhD, Hugh A. Sampson, MD Journal of Allergy and Clinical Immunology Volume 125, Issue 3, Pages 695-702.e6 (March 2010) DOI: 10.1016/j.jaci.2009.12.017 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Comparison of IgE and IgG4 binding frequency to peptides of 5 milk allergens between the different clinical phenotypes. The x-axis shows the overlapping peptides, and the y-axis shows the percentage of patients within each group showing positive binding to each peptide. Binding for IgE is indicated above the x-axis line; binding for IgG4 is below the line. Candidates of informative epitopes are indicated with numbers. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Heat map of IgE binding to candidates of informative epitopes from the allergic subjects compared with the HM-tolerant/outgrown subjects. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Amino acid sequence of milk allergens. Candidates of informative epitopes identified based on the binding frequency are in bold and underlined. Informative epitopes recognized by greater than 50% of the allergic patients with high-affinity IgE antibodies are highlighted in gray. The numbers and letters indicated on top of each epitope correspond to the ones indicated in the other figures. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Heat map showing the effects of competition on 7 allergic and 7 HM-tolerant/outgrown subjects. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Scatter plot comparing IgE binding from 7 allergic (red) and 7 HM-tolerant/outgrown (blue) subjects between the standard protocol (x-axis) and the competition assay (y-axis). Each spot represents IgE binding (represented as a z score) to 1 milk peptide. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Comparison of IgE peptide-binding frequency between groups using the standard protocol and competition assay. Informative epitopes recognized by greater than 50% of the allergic patients with high-affinity IgE antibodies are indicated with letters. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Scatter plot showing the correlation of IgE and IgG4 binding of 8 randomly selected subjects from each group. Each spot represents IgE binding (represented as a z score) to 1 milk peptide. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Distribution of IgE-binding peptides for each group. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effect of temperature. Scatter plot comparing IgE binding between the standard protocol (x-axis) and competition with 2 mg/mL caseins and 3 mg/mL β-lactoglobulin at different temperatures (y-axis) for 4 hours. Each spot represents IgE binding (represented as a z score) to 1 peptide of milk allergens. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effect of competitor concentration Effect of competitor concentration. Scatter plot comparing IgE binding of the serum pool between the standard protocol (x-axis) and competition with a one-third serial dilution of 2 mg/mL caseins and 3 mg/mL β-lactoglobulin (y-axis) at 16°C for 4 hours. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Effect of competition time Effect of competition time. Scatter plot comparing IgE binding of a subject with milk allergy and a subject who had outgrown his or her allergy between the standard protocol (x-axis) and 1 mg/mL caseins and 1.5 mg/mL β-lactoglobulin (y-axis) at 16°C for different competition time. Journal of Allergy and Clinical Immunology 2010 125, 695-702.e6DOI: (10.1016/j.jaci.2009.12.017) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions