ALERBLOT: Interest of 2D Immunoblot in diagnosis of wheat allergy

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ALERBLOT: Interest of 2D Immunoblot in diagnosis of wheat allergy Collaboration Bertholet C1, Quinting B2, Courtois J2, Delahaut P3 and Gadisseur R1. 1: CHU Liège, Belgium, c.bertholet@chu.ulg.ac.be and romy.gadisseur@chu.ulg.ac.be. 2: CRIG Liège, Belgium, b.quinting@helmo.be and j.courtois@crig.be. 3 : CER Group Marloie, Belgium, p.delahaut@cergroup.be. Introduction Wheat allergy is relatively common and related clinical manifestations depend on the involved allergen and the way of exposure: wheat dependent exercise induced anaphylaxis (WDEIA), baker's asthma (BA), atopic dermatitis (AD), pollen rhinitis (PR) and urticaria. Many wheat allergens have been described but due to the wheat protein complexity, only a small number of allergens has been related to a specific allergic manifestation (ω5-gliadin is reported in WDEIA, for example). For the same reason, sensitizations cannot be detected by traditional diagnostic tests given that only a few molecular allergens are available, in addition to wheat protein extract : gluten, gliadins, Tri a 19 allergen (ω5-gliadin) and Tri a 14 (LTP) . Our study aims to use the protein separation capacity of 2D Immunoblot to contribute to wheat allergy diagnosis by comparing sensitization profile of patients with common symptomatology. Materials and methods Fig 1 : 2D electrophoresis gel of total wheat protein extract. Standardized protein extracts from wheat seeds were separated on the basis of their isoelectric point and their size. We selected 169 patients sensitized to wheat on the basis of their positive specific IgE (sIgE) to wheat. Amongst the 169 sera, 25 sera were classified into 4 distinct groups following symptomatology : WDEIA, AD, PR, and mixed AD/PR. All of them were analyzed on 2D Immunoblot performed with the standardized wheat seeds extracts. The sIgE sensitization profiles of the four patient groups were compared. Fig 2 : 2D Immunoblot with pooled sera of of wheat allergic patients. Results : Comparison of sensitization profiles related on clinical symptoms Hypothesis for the identification of wheat allergens based on isoelectric points and molecular masses available in allergens data bases. 1. WDEIA 2. Pollen rhinitis (PR) Fig 3 : Superposition of 2D immunoblot of WDEIA patients : case 1 (black), case 2 (blue), case 3 (yellow) and case 4 (red). Serpin (Tri a 33) ? ω5-gliadin (Tri a 19) ? - Tri a 20 - Tri a 27 - Tri a 31 - Tri a 32 Tri a 19 ? Fig 4 : Superposition of 2D immunoblot of PR patients : case 15 (pink), case 16 (black), case 17 (green), case 18 (red), case 19 (dark blue), case 20 (yellow) and case 21 (light blue). ? ? 3. Atopic dermatitis (DA) -Tri a TLP Tri a 12 Tri a 36 Tri a 40 Tri a 42 ? Tri a 26 ? Tri a 19 ? Tri a 14 Tri a 44 Tri a 45 Tri a 7k-LTP Fig 6 Superposition of 2D immunoblot of DA patients both subgroups (DA1 and DA2. Fig 5 : Superposition of 2D immunoblot of DA 1 patients : case 5 (black), case 8 (red), case 10 (green), case 12 (yellow), case 13 (blue) and case 14 (orange). Fig 6 : Superposition of 2D immunonlot of DA 2 patients : case 6 (red), case 7 (yellow), case 9 (blue) and case 11 (black). ? ? Conclusions References At this stage, sensitization profiles obtained for the 3 symptomatic groups identified different specific protein areas (red circles). These areas recognized by sIgE concern one or more allergens. The identification of this specific protein spots is currently under investigation by LC-MS/MS mass spectrometry. 1. Inomata, N. (2009). "Wheat allergy." Curr Opin Allergy Clin Immunol 9(3): 238-243. 2. Beaudoin, E., Renaudin, J.M., et al. (2007). “Wheat allergy in adults”. Rev Fr Allergol 47: 175-179. 3. Battais F, Richard C, Jacquenet S, Denery-Papini S, Moneret-Vautrin D.A. Wheat grain allergies: an update on wheat allergens. Eur Ann Allergy Clin Immunol 2008;40:67-76. 4. Allergome, http://www.allergome.org/. 5. Allergen, http://www.allergen.org/.