Peanut epitopes for IgE and IgG4 in peanut-sensitized children in relation to severity of peanut allergy Annebeth E. Flinterman, MD, Edward F. Knol,

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Peanut epitopes for IgE and IgG4 in peanut-sensitized children in relation to severity of peanut allergy Annebeth E. Flinterman, MD, Edward F. Knol, PhD, Doerthe A. Lencer, MD, Ludmilla Bardina, MS, Constance F. den Hartog Jager, BSc, Jing Lin, PhD, Suzanne G.M.A. Pasmans, MD, PhD, Carla A.F.M. Bruijnzeel-Koomen, MD, PhD, Hugh A. Sampson, MD, Els van Hoffen, PhD, Wayne G. Shreffler, MD, PhD Journal of Allergy and Clinical Immunology Volume 121, Issue 3, Pages 737-743.e10 (March 2008) DOI: 10.1016/j.jaci.2007.11.039 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IgE and IgG4 peptide binding to peanut allergen–derived peptides is significantly greater in sera from peanut-sensitized patients versus sera from control subjects. IgE SNR versus IgG4 SNR is shown. Dashed lines indicate cutoffs (see the Methods section). Numbers indicate percentages above cutoff. A, Control sera (n = 6). B, Peanut-sensitized sera at baseline (n = 24). Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Multiple sequential epitope-rich regions within the peanut allergens Ara h 1 through Ara h 3. A, Representation of mean log SNR for binding of patient IgE (blue line, n = 24) and IgG4 (green line, n = 24) versus control (red lines, n = 6). Dashed lines indicate cutoff values (see the Methods section and Fig E2). B, Heat map image of individual IgE/IgG4 SNR showing diversity of individual binding patterns (see the Methods section for details). Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Greater IgE epitope recognition corresponds to greater clinical sensitivity. The distribution of reaction scores is shown for each quartile of patients binned into quartiles according to the number of positive IgE epitopes. Boxes represent median and 25% to 75% distribution, whiskers represent range, and solid circles represent individual reaction scores. Patients in the highest quartile for IgE epitope number (quartile 4, n = 6), had significantly higher combination scores in comparison with those with the least IgE binding (quartile 1, n = 6). ∗P = .021. Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Clinical sensitivity corresponds to greater IgE epitope recognition. IgE versus IgG4 SNR is shown in separate plots by quartiles of clinical sensitivity (A to D for least to most sensitive; see the Methods section). Dashed lines represent cutoffs. Numbers in each quadrant represent percentage above cutoff. Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 IgE and IgG4 binding patterns are largely stable over 20 months. Correspondence between baseline and 20-month follow-up is shown as a topographic representation of the number of patients with positive results for each peptide before (x-axis) and 20 months after (y-axis) DBPCFC (n = 22). A, IgE; B, IgG4. The diagonal represents identity between t=0 and t=20 months. Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Representation of mean log IgE SNR of raw data by array element before near-neighbor normalization to remove false-positive signal. Patient IgE is shown by the blue line (n = 24), and control is shown by the red line (n = 6). See Fig 2 for comparison with data after near-neighbor correction. CV, Coefficient of variation. Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Distribution of signal from control sera (n = 6) and determination of cutoff. Histograms of IgE (A) and IgG4 (B) SNR for peptide array elements incubated with control sera from patients without peanut allergy are shown. The vertical broken line represents the cutoff point at a P value of .995 by using the formula p(k)=(k−1/3)/(n+1/3), which is used for determination of “positive” signal. See Fig 1 for comparison of signal from control and patient sera. Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Correlation between peanut-specific IgE levels and the number of epitopes recognized by IgE per subject (r = 0.579). Journal of Allergy and Clinical Immunology 2008 121, 737-743.e10DOI: (10.1016/j.jaci.2007.11.039) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions