In vivo allergenic activity of a hypoallergenic mutant of the major fish allergen Cyp c 1 evaluated by means of skin testing Nikolaos Douladiris, MD,

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In vivo allergenic activity of a hypoallergenic mutant of the major fish allergen Cyp c 1 evaluated by means of skin testing Nikolaos Douladiris, MD, Birgit Linhart, PhD, Ines Swoboda, PhD, Antonia Gstöttner, MSc, Emilia Vassilopoulou, PhD, Frank Stolz, PhD, Rudolf Valenta, MD, Nikolaos G. Papadopoulos, MD Journal of Allergy and Clinical Immunology Volume 136, Issue 2, Pages 493-495.e8 (August 2015) DOI: 10.1016/j.jaci.2015.01.015 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Reduced allergenic activity of mCyp c 1 compared with wtCyp c 1 demonstrated by means of skin testing of patients with fish allergy. Twelve patients were pricked with increasing concentrations (x-axis: 1, 4, 16, and 32 μg/mL) of wtCyp c 1 (wt) and mCyp c 1 (m). Displayed are box plots representing wheal areas (y-axis: millimeter squared) for patients, with medians indicated by horizontal lines and outliers represented by asterisks (∗). Significant differences (***P < .001) between wtCyp c 1 and mCyp c 1 are displayed for each tested concentration. Journal of Allergy and Clinical Immunology 2015 136, 493-495.e8DOI: (10.1016/j.jaci.2015.01.015) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 IgE sensitization profile of the children with fish allergy determined by using ISAC. Sera from 8 of the patients were tested for IgE reactivity to 103 different allergens by using ISAC. The number of patients (y-axis) displaying IgE reactivity to the allergens (x-axis) is shown. Journal of Allergy and Clinical Immunology 2015 136, 493-495.e8DOI: (10.1016/j.jaci.2015.01.015) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Correlation of Cyp c 1–specific IgE levels measured by using ImmunoCAP (x-axis) and ISAC (y-axis). Journal of Allergy and Clinical Immunology 2015 136, 493-495.e8DOI: (10.1016/j.jaci.2015.01.015) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 IgE reactivity to natural fish allergens. Nitrocellulose-blotted natural allergen extracts from carp (A), cod (B), and tuna (C) were incubated with sera from 12 children with fish allergy (1-12; Co, positive control serum from a patient with fish allergy), serum from a nonallergic subject (N), or buffer alone (B). Bound IgE was detected with iodine125–labeled anti-IgE antibodies and visualized by means of autoradiography. Molecular weights (in kilodaltons) are displayed on the left margin. Journal of Allergy and Clinical Immunology 2015 136, 493-495.e8DOI: (10.1016/j.jaci.2015.01.015) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 A, SDS-PAGE of purified recombinant wild-type Cyp c 1 (wt) and mutant Cyp c 1 (mu). A molecular weight marker (M; in kilodaltons) is shown on the left side. B, Comparison of dot-blotted wtCyp c 1 (upper part) with mCyp c 1 (lower part) regarding IgE reactivity as tested with sera from allergic patients (Co and 1-12), serum from a nonallergic subject (N), or buffer alone (B). Journal of Allergy and Clinical Immunology 2015 136, 493-495.e8DOI: (10.1016/j.jaci.2015.01.015) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Dot-blotted wtCyp c 1 and mCyp c 1 were tested regarding IgE reactivity with sera from patients with fish allergy (Co and 13-27), serum from a nonallergic subject (N), or buffer alone (B). IgE was detected with an iodine125–labeled anti-IgE antibody, and counts per minute values corresponding to wtCyp c 1– and mCyp c 1–bound IgE, as well as the percentage reduction of IgE binding to mCyp c 1, are shown. Journal of Allergy and Clinical Immunology 2015 136, 493-495.e8DOI: (10.1016/j.jaci.2015.01.015) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions