Visualization of clustered IgE epitopes on α-lactalbumin

• Published in: Journal of allergy and clinical immunology Vol. 125; no. 6; pp. 1279 - 1285.e9
• Main Authors: Hochwallner, Heidrun, MSc, Schulmeister, Ulrike, PhD, Swoboda, Ines, PhD, Focke-Tejkl, Margit, PhD, Civaj, Vera, Balic, Nadja, Nystrand, Mats, PhD, Härlin, Annika, BSc, Thalhamer, Josef, PhD, Scheiblhofer, Sandra, PhD, Keller, Walter, PhD, Pavkov, Tea, PhD, Zafred, Domen, MSc, Niggemann, Bodo, MD, Quirce, Santiago, MD, Mari, Adriano, MD, Pauli, Gabrielle, MD, Ebner, Christof, MD, Papadopoulos, Nikolaos G., MD, Herz, Udo, PhD, van Tol, Eric A.F., PhD, Valenta, Rudolf, MD, Spitzauer, Susanne, MD
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01-06-2010; Elsevier
• Subjects: Allergen; Allergic diseases; Allergy and Immunology; Animals; Biological and medical sciences; Cattle; Cells, Cultured; Circular Dichroism; Cloning, Molecular; Digestive allergic diseases; Epitopes, B-Lymphocyte - chemistry; Epitopes, B-Lymphocyte - metabolism; Escherichia coli - genetics; Feasibility Studies; food allergy; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Histamine Release - immunology; Humans; IgE epitope; Immunoglobulin E - blood; Immunoglobulin E - immunology; Immunopathology; Lactalbumin - genetics; Lactalbumin - immunology; Lactalbumin - isolation & purification; Lactalbumin - metabolism; Mass Spectrometry; Medical sciences; Microarray Analysis; milk allergy; Milk Hypersensitivity - blood; Milk Hypersensitivity - diagnosis; Milk Hypersensitivity - immunology; Peptide Fragments - chemistry; Peptide Fragments - metabolism; recombinant allergen; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sarcoidosis. Granulomatous diseases of unproved etiology. Connective tissue diseases. Elastic tissue diseases. Vasculitis; National Institutes of Health; recombinant allergen; Cow's milk; Isopropyl-β-D-thiogalactopyranoside; HSA; rα-La; Ethyleneglycol-bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid; RBL; α-La; CD; Human serum albumin; nα-La; Natural α-lactalbumin; CM; NIH; food allergy; Recombinant α-lactalbumin; milk allergy; Circular dichroism; EGTA; α-Lactalbumin; Rat basophil leukemia; PBST; IgE epitope; IPTG; PBS with 0.5% vol/vol Tween 20; Allergen; Food allergy; Immunopathology; Immunology; Antigenic determinant; Lactalbumin; IgE; Digestive diseases; Recombinant protein
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/j.jaci.2010.03.007

Background α-Lactalbumin (α-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. Objective We performed molecular, structural, and immunologic characterization of α-La. Methods Recombinant α-lactalbumin (rα-La) was expressed in Escherichia coli , purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. Results According to circular dichroism analysis, rα-La represented a folded protein with a high thermal stability and refolding capacity. rα-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. rα-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of α-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. Conclusions rα-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.