Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library Brigitte Hantusch, MSc, Sigurd Krieger, TA, Eva Untersmayr,

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Presentation transcript:

Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library Brigitte Hantusch, MSc, Sigurd Krieger, TA, Eva Untersmayr, MD, Isabella Schöll, MSc, Regina Knittelfelder, Sabine Flicker, PhD, Susanne Spitzauer, MD, Rudolf Valenta, MD, George Boltz-Nitulescu, PhD, Otto Scheiner, PhD, Erika Jensen-Jarolim, MD Journal of Allergy and Clinical Immunology Volume 114, Issue 6, Pages 1294-1300 (December 2004) DOI: 10.1016/j.jaci.2004.06.048 Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 1 A, Phylogenetic tree indicating the degree of similarity between the selected peptides. Identical residues are boxed. B, Alignment of peptides with the amino acid sequences of Phl p 5a and Phl p 6. Identical residues are indicated in bold, and physicochemically related amino acids are indicated in bold-faced italics. Additionally, identical and related amino acids are boxed in gray. Predicted α-helices are underlined. Journal of Allergy and Clinical Immunology 2004 114, 1294-1300DOI: (10.1016/j.jaci.2004.06.048) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 2 Molecular modeling and IgE epitope mapping on the 2 AR repeats of Phl p 5a. A and B, Ribbon diagrams visualizing the AR repeats. C and D, Molecular surface representations. Alignment regions with the peptides (designations correspond to clone numbers in Table I) are highlighted in red (AR repeat 1) and blue (AR repeat 2), respectively. E and F, One hundred eighty degree rotation of the 2 domains. Journal of Allergy and Clinical Immunology 2004 114, 1294-1300DOI: (10.1016/j.jaci.2004.06.048) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 3 Phage-displayed peptides bind specifically to a monoclonal Phl p 5a–reactive Fab fragment (black columns) and to Phl p 5a IgE (white columns) but not to the isotype controls PS- and SUS11-IgE (gray columns). Twelve individual phage clones (1 × 108 cfu/mL) and WT-phage lacking an insert were added to the coated wells. The data represent the mean ± SD of 4 individual assays. Journal of Allergy and Clinical Immunology 2004 114, 1294-1300DOI: (10.1016/j.jaci.2004.06.048) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 4 A-C, Phage clones of the acidic (1) and competitive (2) elution method and a phage cocktail (3) tested for IgE specificity in ELISA. D-F, BIAcore sensorgrams of Phl p 5–specific IgE binding to Phl p 5a. G-I, Bet v 1–specific IgE binding to Bet v 1 in the absence of phages (IgE), in the presence of WT-phage, and in the presence of phage clones. One typical sensorgram of 3 individual measurements is shown. Journal of Allergy and Clinical Immunology 2004 114, 1294-1300DOI: (10.1016/j.jaci.2004.06.048) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 5 IgG reactivity of Biozzi mice immune sera elicited by phagotopes F1, F29, H32, and WT-phage toward Phl p 5a (A) and Bet v 1 (B). The x-axis shows the time schedule of blood taking. The y-axis shows mean IgG reactivities and SDs of mouse groups (n=5), testing mice individually. ∗∗P < .05 for F1- and F29-phage immune sera on day 35 compared with preimmune sera and WT-phage sera on day 35. Journal of Allergy and Clinical Immunology 2004 114, 1294-1300DOI: (10.1016/j.jaci.2004.06.048) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions