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IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3  Merima Bublin, PhD, Maria Kostadinova,

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Presentation on theme: "IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3  Merima Bublin, PhD, Maria Kostadinova,"— Presentation transcript:

1 IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3  Merima Bublin, PhD, Maria Kostadinova, MSc, Christian Radauer, PhD, Christine Hafner, MD, Zsolt Szépfalusi, MD, Eva-Maria Varga, MD, Soheila J. Maleki, PhD, Karin Hoffmann-Sommergruber, PhD, Heimo Breiteneder, PhD  Journal of Allergy and Clinical Immunology  Volume 132, Issue 1, Pages e12 (July 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 IgE cross-reactivity of Ara h 1, Ara h 2, and Ara h 3 tested by using an IgE ELISA inhibition assay. Cross-inhibitions were tested in a dose-dependent manner with sera from 10 patients with peanut allergy, and values are expressed as maximum inhibition (A) and IC50 (B). Median values (solid lines) with interquartile ranges are shown. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Sequence comparison of Ara h 2 (A) and Ara h 6 (B) with Ara h 1 and Ara h 3. The positions of the synthetic peptides and of Ara h 2 and Ara h 6 peptide-matching sequences from Ara h 1 or Ara h 3 are indicated by black bars. Residue numbers refer to numbering of UniProt entries Q6PSU2 and Q647G9, which include signal peptides. Occurrences of the repetitive sequence DPYSPS are boxed, and α-helices are shaded. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 IgE reactivity profiles of 10 patients with peanut allergy to peanut allergens and synthetic peptides. IgE binding was determined by using ELISA. The intensity of IgE binding is represented by weighted OD (405 nm) values in a grading scale ranging from less than 0.1 (white cells) to 0.8 or greater (black cells). Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Inhibition of IgE binding to the Ara h 2–derived peptides AH2-1, AH2-3a, and AH2-3c by peanut allergens. IgE inhibition was performed with individual sera containing IgE specific to at least 1 of the tested peptides. The graphs represent the median values of inhibitions obtained with individual sera. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Dose-dependent inhibition ELISA of IgE binding to Ara h 1 to Ara h 3 with IgE-binding, Ara h 2–derived peptides. Binding of IgE from 5 individual sera (P1-P5) was inhibited with 10-fold serial dilutions of a mixture of peptides AH2-1, AH2-3a, and AH2-3c. Negative controls performed with all sera preincubated with 10-fold serial dilutions of peptides AH2-2 and AH2-4 are shown as average values with SEMs. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 IgE cross-reactivity of Ara h 1, Ara h 2, and Ara h 3 tested by using an IgE ELISA inhibition assay. Cross-inhibitions were performed in a dose-dependent manner with 10 sera from patients with peanut allergy, and values are expressed as the percentage of IgE inhibition. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 IgE cross-reactivity of Ara h 1, Ara h 2, and Ara h 3 tested by using an IgE ELISA inhibition assay. Cross-inhibitions were performed in a dose-dependent manner with 10 sera from patients with peanut allergy, and values are expressed as the percentage of IgE inhibition. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 IgE cross-reactivity of Ara h 1, Ara h 2, Ara h 3, and Ara h 6 tested by using an IgE ELISA inhibition assay. Cross-inhibitions were tested in a dose-dependent manner with 5 sera from patients with peanut allergy. Median values (solid lines) with interquartile ranges are shown. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 A, Circular dichroism spectra of natural Ara h 2 and rAra B, Inhibitions of IgE binding to Ara h 1, Ara h 2, and Ara h 3 by rAra h 2. One microgram per milliliter of allergen was coated to microtiter plates, and binding of IgE from sera of patients with peanut allergy was inhibited by 10 μg/mL rAra h 2. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 A, Analysis of purity of Ara h 1, Ara h 2, and Ara h 3 by using immunoblotting with polyclonal antibodies raised against individual allergens. The left panel shows Commassie-stained Ara h 1, Ara h 2, and Ara h 3 (2 μg per lane). B, IgE cross-reactivity of Ara h 1, Ara h 2, and Ara h 3 tested by using IgE immunoblotting with sera from patients with peanut allergy sera. The panels represent IgE binding and cross-inhibitions by each of the 3 allergens. Inhibitions were performed with 10 μg/mL of the inhibiting allergen. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E5 A, Positions of the synthetic peptides used in this study on the sequence of Ara h Alignment of peptide sequences obtained by comparing Ara h (B) and Ara h (C) with Ara h 1 and Ara h 3. Residues identical between Ara h 2 or Ara h 6 and Ara h 1 or Ara h 3 are shown in boldface. UniProt accession numbers are as follows: Ara h , Q6PSU2; Ara h , Ara h , Q647G9; P43238; Ara h 1-P17, P43237; Ara h 1, E5G076; Ara h 3.0101, O82580; Ara h , Q9SQH7; iso-Ara h 3, Q0GM57; Ara h 3-Ahy-1, Q647H4; Ara h 3-Ahy-2, Q647H3; Ara h 3-Ahy-3, Q647H2; and Ara h 3-Ahy-6, A1DZF0. All residue numbers refer to numberings of the original UniProt entries that include signal peptides. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E6 Localization of putative cross-reactive peptides on the structures of Ara h 1 (PDB accession no.: 3smh; A),E1 Ara h 2 (3ob4; B), Ara h 3 (3c3v; C),E2 and Ara h 6 (1w2q; D).E3 Nonstructured loops missing in the crystal structures were added with Modeller 9v3.E4 Graphics were generated with UCSF Chimera.E5 Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E7 Inhibition of IgE binding to Ara h 2–derived peptides by peanut allergens. IgE inhibition of individual sera (P1-10) containing IgE specific for at least 1 of the tested peptides was performed with 0.01, 0.1, and 1 nmol/L of the allergen or peptide. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 Fig E7 Inhibition of IgE binding to Ara h 2–derived peptides by peanut allergens. IgE inhibition of individual sera (P1-10) containing IgE specific for at least 1 of the tested peptides was performed with 0.01, 0.1, and 1 nmol/L of the allergen or peptide. Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions


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