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- Curin, M, et al.
(författare)
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Clustering of conformational IgE epitopes on the major dog allergen Can f 1
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1, s. 12135-
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Tidskriftsartikel (refereegranskat)abstract
- Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients’ IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies.
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- Apostolovic, D, et al.
(författare)
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Peptidomics of an in vitro digested α-Gal carrying protein revealed IgE-reactive peptides
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1, s. 5201-
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian carbohydrate galactose-α1,3-galactose (α-Gal) causes a novel form of food allergy, red meat allergy, where patients experience severe allergic reactions several hours after red meat consumption. Here we explored gastric digestion of α-Gal glycoproteins using an in vitro model. Bovine thyroglobulin (BTG), a typical α-Gal carrying glycoprotein, was digested with pepsin. The resulting peptides were characterised by SDS PAGE, immunoblot and ImmunoCAP using sera from 20 red meat allergic patients. During pepsinolysis of BTG, a wide range of peptide bands was observed of which 14 to 17 kDa peptides remained stable throughout the gastric phase. The presence of the α-Gal epitope on the obtained peptides was demonstrated by an anti-α-Gal antibody and IgE from red meat allergic patients. The α-Gal digests were able to inhibit up to 86% of IgE reactivity to BTG. Importantly, basophil activation test demonstrated that the allergenic activity of BTG was retained after digestion in all four tested patients. Mass spectrometry-based peptidomics revealed that these peptides represent mostly internal and C-terminal parts of the protein, where the most potent IgE-binding α-Gal residues were identified at Asn1756, Asn1850 and Asn2231. Thus allergenic α-Gal epitopes are stable to pepsinolysis, reinforcing their role as clinically relevant food allergens.
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