Component resolution reveals additional major allergens in patients with honeybee venom allergy Julian Köhler, MS, Simon Blank, PhD, Sabine Müller, MD,

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Component resolution reveals additional major allergens in patients with honeybee venom allergy Julian Köhler, MS, Simon Blank, PhD, Sabine Müller, MD, Frank Bantleon, DiplBiol, Marcel Frick, MS, Johannes Huss-Marp, MD, Jonas Lidholm, PhD, Edzard Spillner, PhD, Thilo Jakob, MD Journal of Allergy and Clinical Immunology Volume 133, Issue 5, Pages 1383-1389.e6 (May 2014) DOI: 10.1016/j.jaci.2013.10.060 Copyright © 2014 The Authors Terms and Conditions

Fig 1 IgE immunoreactivity of individual patient sera with recombinant allergens. IgE reactivity to HBV allergens of sera from patients with HBV allergy (n = 144), patients with YJV allergy (n = 40), and HBV-exposed but nonallergic control subjects (NA; n = 40). The lower-end cutoff of the CAP-FEIA (<0.35 kUA/L) is represented as a dotted line. Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions

Fig 2 Diagnostic sensitivity of sIgE to different combinations of HBV allergens. Detection of IgE reactivity to a panel of HBV allergens increases diagnostic sensitivity in patients with HBV allergy (n = 144). Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions

Fig 3 IgE reactivity to single HBV allergens in relation to sIgE to HBV. A, IgE reactivity to HBV (ImmunoCAP i1) in patients with HBV allergy (n = 144) in relation to the sum of IgE reactivity to Api m 1, Api m 2, Api m 3, Api m 4, Api m 5, Api m 10, and CCDs. B, The relative sIgE reactivity to single HBV allergens was calculated as a ratio of sIgE reactivity to HBV and displayed as a whisker plot with medians; 5th, 25th, 75th, and 95th percentiles; and outliers. C, The median was used to present the relative contribution to IgE reactivity of single allergens as a pie chart. Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions

Fig 4 Inhibition of IgE reactivity to HBV (ImmunoCAP i1) by single HBV allergens or crude or therapeutic HBV preparations. A, CAP-FEIA inhibition of sIgE reactivity to HBV (ImmunoCAP i1) was performed with Api m 1 or Api m 10 as inhibitors at increasing concentrations in patients with predominant Api m 1 sensitization (Api m 1+ Api m 10−; n = 3, mean ± SD) or in patients with a predominant Api m 10 sensitization (Api m 1− Api m 10+; n = 3, mean ± SD). B, CAP-FEIA inhibition of sIgE reactivity to HBV (ImmunoCAP i1) was performed with Api m 1, Api m 3, or Api m 10 at 300 nmol/L in patients with HBV allergy (n = 36). The degree of CAP-FEIA inhibition was correlated with the relative IgE reactivity to Api m 1, Api m 3, or Api m 10, as calculated in Fig 3 (P < .0001, r = .8082). C, CAP-FEIA inhibition of sIgE reactivity to HBV (ImmunoCAP i1) was performed with a crude HBV, a diagnostic HBV, or 2 different therapeutic HBV preparations at 300 μg/mL in patients with predominant Api m 1 sensitization (Api m 1+ Api m 10−; n = 4, mean ± SD) or in patients with predominant Api m 10 sensitization (Api m 1− Api m 10+; n = 4, mean ± SD). BV, Bee venom. Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions

Fig 5 Induction of allergen-specific IgG4 during HBV immunotherapy. sIgG4 responses to Api m 1, Api m 3, Api m 4, and Api m 10 were analyzed in patients before and 12 to 36 months after initiation of HBV immunotherapy (n = 20). The induction of sIgG4 was expressed as the ratio of sIgG4 during immunotherapy/sIgG4 before immunotherapy and is displayed as whisker plots with medians; 5th, 25th, 75th, and 95th percentiles; and outliers. Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions

Fig E1 Recombinant expression and immunoreactivity of venom allergens. SDS-PAGE analyses of the purified recombinant allergens Api m 1, Api m 2, Api m 3, Api m 5, and Api m 10, as visualized by using Coomassie blue staining. Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions

Fig E2 Relationship between levels of sIgE to HBV and the number of different HBV allergens recognized in HBV-monosensitized (A) and HBV and YJV double-sensitized patients (B). Journal of Allergy and Clinical Immunology 2014 133, 1383-1389.e6DOI: (10.1016/j.jaci.2013.10.060) Copyright © 2014 The Authors Terms and Conditions