Identification of an immunodominant IgE epitope of Der f 40, a novel allergen of Dermatophagoides farinae

Background: House dust mites (HDMs), including Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) species, represent a major source of inhalant allergens that induce IgE-mediated anaphylactic reactions. HDM allergen identification is important to the diagnosis and treatment of allergic diseases. Here, we report the identification of a novel HDM allergen, which we suggest naming Der f 40, and its immunodominant IgE epitopes. Methods: The recombinant protein Der f 40 was expressed using a pET prokaryotic expression system and purified with Ni-NTA resins. IgE binding activity was evaluated by IgE-western blot, dot-blot, and ELISA. Mast cell activation testing was performed to assess the cellular effects of IgE binding in mouse bone marrow derived mast cells (BMMCs) expressing human Fc & epsilon;RI. IgE binding assays were performed with truncated and hybrid Der f 40 protein molecules to find immunodominant IgE epitopes. Results: A 106-amino acid (aa) recombinant Der f Group 40 protein (rDer f 40) was obtained (GenBank accession No. XP_046915420.1) as thiredoxin-like protein. Der f 40 was shown to bind IgE from HDM allergic serum in vitro (9.68%; 12/124 in IgE-ELISA), and shown to promote the release of b-hexosaminidase from BMMCs dose-dependently when administered with HDM allergic sera. The Der f Group 40 protein was named Der f 40 and listed in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. IgE binding assays with Der f 40-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The 76-106-aa region of C-terminus was identified as an immunodominant IgE epitope of Der f 40. Conclusion: A novel HDM allergen with robust IgE binding activity was identified and named Der f 40. An immunodominant IgE epitope of Der f 40 with conformational dependency was identified in the C-terminus (aa 76-106). These findings provide new information that may be useful in the development of diagnostic and therapeutic agents for HDM allergy.