Patients Allergic to Fish Tolerate Ray Based on the Low Allergenicity of Its Parvalbumin Tanja Kalic, MSc, Francoise Morel-Codreanu, MD, Christian Radauer,

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Patients Allergic to Fish Tolerate Ray Based on the Low Allergenicity of Its Parvalbumin Tanja Kalic, MSc, Francoise Morel-Codreanu, MD, Christian Radauer, PhD, Thimo Ruethers, MSc, Aya C. Taki, PhD, Ines Swoboda, PhD, Christiane Hilger, PhD, Karin Hoffmann-Sommergruber, PhD, Markus Ollert, MD, DMSc, Christine Hafner, MD, Andreas L. Lopata, PhD, Martine Morisset, MD, PhD, Heimo Breiteneder, PhD, Annette Kuehn, PhD The Journal of Allergy and Clinical Immunology: In Practice Volume 7, Issue 2, Pages 500-508.e11 (February 2019) DOI: 10.1016/j.jaip.2018.11.011 Copyright © 2018 The Authors Terms and Conditions

Figure 1 IgE levels to α-parvalbumins are significantly lower than IgE levels to β-parvalbumins. A, IgE titers (kUA/L) to α-parvalbumins and β-parvalbumins among 18 patients allergic to fish were determined by ELISA. B, Patient-specific IgE recognition patterns to α-parvalbumins and β-parvalbumins. The signal intensity of parvalbumin-specific IgE is represented in a grading log10 scale for concentrations of measured parvalbumin-specific IgE (kUA/L). The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure 2 IgE antibody cross-reactivity of cod β-parvalbumin to salmon (β) and ray (α) parvalbumin determined by inhibition ELISA in patients allergic to bony fish (n = 10). Microtiter plates were coated with cod β-parvalbumin and sera preincubated with cod β-parvalbumin (A), salmon β-parvalbumin (B), or ray α-parvalbumin (C) at increasing concentrations. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Basophil response to α-parvalbumins was lower than to β-parvalbumins. Data indicate basophil activation (measured as percentage of CD63+ basophils) in response to stimulation with different doses of fish parvalbumins in patients allergic to fish (n = 18). The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure 4 α-Parvalbumins show lower capacity than β-parvalbumins to induce skin reactions in patients allergic to fish. SPTs were performed with different doses of natural purified fish parvalbumins. Parvalbumins were used in concentrations ranging from 0.1 μg/mL to 50 μg/mL. Average wheal diameter of greater than or equal to 3 mm compared with negative control was rated as a positive response. (X) indicates that SPT was not performed. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure E1 Diagnostic flowchart for assessing tolerance of ray. Numbers in parentheses represent the numbers of subjects for each step. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure E2 Gating strategy in BAT. Basophilic cells were selected from whole blood based on CCR3high/SSClow. Activation of basophils was determined by expression of an activation marker CD63. Expression of CD63 for unstimulated control, 2 stimulation controls (anti-FCεRI mAb and fMLP), and stimulation with 100 ng/mL cod parvalbumin is demonstrated for P1 as an example. fMLP, Formyl-methionyl-leucyl-phenylalanine. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure E3 Characterization of parvalbumins from bony and cartilaginous fish species. A, Coomassie-stained SDS-PAGE gel of purified parvalbumins. B, Western blot using commercial antiparvalbumin antibodies. C, Circular dichroism (CD) spectra of purified parvalbumins. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure E4 BAT. Percentages of CD63+ basophils (y-axes) at different concentrations of β-parvalbumins and α-parvalbumins are represented for individual subjects allergic to fish (P1-P18). The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure E5 Response to positive (anti-FcεRI and fMLP) and negative (stimulation buffer) controls in BAT for subjects with fish allergy. Ctrl, Control; fMLP, formyl-methionyl-leucyl-phenylalanine; Neg, negative; pos, positive. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions

Figure E6 Detection of fish allergens in ray extract. A, Coomassie-stained SDS-PAGE of extract of raw ray filet and ray filet cooked for 5, 10, or 20 minutes. B-D, Immunoblots with ray extract using antiparvalbumin antibody (Fig E6, B), antienolase antibody (Fig E6, C), and antialdolase antibody (Fig E6, D). E, IgE immunoblots using extract of raw ray filet. P, E, and A stand for controls parvalbumin, enolase and aldolase, respectively. P4 and P6 were additionally tested on extract of cooked ray (4* and 6*). Ab, Antibody. The Journal of Allergy and Clinical Immunology: In Practice 2019 7, 500-508.e11DOI: (10.1016/j.jaip.2018.11.011) Copyright © 2018 The Authors Terms and Conditions