Identificação de isoformas antigênicas de Dermatophagoides pteronyssinus reconhecidas por anticorpos IgE e IgG1 em pacientes atópicos e não atópicos

Abstract

The house dust mite Dermatophagoides pteronyssinus (Dp) is a major source of indoor allergens in the world and considered important for sensitization of genetically predisposed subjects. Some allergens from this mite can occur as isoallergens with differences in amino acid sequences or glycosylation, leading to differences in allergenic sensitization. The aim of this study was to investigate new immunodominants antigens with potential application for the laboratorial diagnosis and monitoring of atopic patients under allergen-specific immunotherapy. Dp extract was separated by two-dimensional (2D) electrophoresis with subsequent immunoblotting for determination of specific IgE and IgG1 binding components. Pooled human sera tested in 2D immunoblotting were chosen from a panel of sera positive to Dp allergens determined by 1D immunoblotting for specific IgE and IgG1 in atopic patients (A) and IgG1 in non-atopic subjects (NA). Patients were selected by skin prick test (SPT) and levels of specific IgE and IgG1 by ELISA. Levels of IgE to Dp and seropositivity were higher in atopics (median EI = 3.8; 100%) than non-atopics (median EI = 0.7; 0%) (P < 0.0001), while levels of Dp-specific IgG1 showed no significant difference between the groups (median EI = 2.9; 84% vs median EI = 2.5; 86%). The major IgE-binding components determined by 1D immunoblotting were the 15 kDa, 60 kDa and 103 kDa bands, and the major IgG1-binding allergens were bands >56 kDa. 2D gel electrophoresis of Dp extract showed a resolution of more than 70 spots throughout the separation range of isoelectric point (pI) 3-10 and molecular size of 11-110 kDa. 2D immunoblotting revealed differences in IgE- and IgG1-binding patterns, with several spots recognized by IgE antibodies ranging from 15 to 103 kDa and pI 5.1-8.0. For IgG1 reactivity in atopic patients, strongly recognized spots were observed at ranges of higher molecular masses (70-103 kDa) and lower pI (5.1- 6.6). For IgG1 reactivity in non-atopic subjects, the most strongly recognized spots were observed ranging from 85 to 103 kDa (pI 5.1-5.8). 2D electrophoresis with subsequent immunoblotting allowed the identification of specific isoallergens recognized by different antibody isotypes in atopic and non-atopic subjects. This information can be used to improve the development of potential candidates for using in diagnostic as well as in monitoring of the levels of specific IgE and IgG1 in atopic patients under immunotherapy.