D2.182 - Mass Spectrometric Identification of Allergens from Dermatophagoides spp. and Blomia tropicalis in Native, Depigmented and Depigmented-Polymerized Extracts

The presence of allergens in extracts used for immunotherapy is essential to guarantee their efficacy. In the case of modified allergenic extracts, allergen identification through immunoassays is not possible. Additionally, immunoassays are not available for all the allergens described. For these reasons, the use of alternative methods such as mass spectrometry is necessary. The aim of this study was to identify allergens present in native, depigmented and depigmented-polymerized extracts of different mite species using mass spectrometry.

Lyophilized samples of native, depigmented and depigmented-polymerized extracts from Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis (LETI Pharma S.L.U.) were solubilized and digested with trypsin. The resulting peptide mixture was analyzed using a Vanquish Neo IFC liquid chromatograph coupled to an Q Exactive HF (Thermo) mass spectrometer. Allergen identification was performed using a database combining allergen sequences described in the International Union of Immunological Societies (IUIS) database with UniProt.

For D. pteronyssinus, 31 allergen sequences are in the IUIS database. Of these, 25 allergens were identified in native extracts, 21 in depigmented extracts and 23 in depigmented-polymerized extracts. For D. farinae, the IUIS database lists 39 allergen sequences, with 31 allergens identified in  native extracts, 30 in depigmented extracts and 29 in depigmented-polymerized extracts. Finally, the IUIS database lists 23 allergen sequences for B. tropicalis, of which 17 were found in native extracts, 19 in depigmented extracts and 16 in depigmented-polymerized extracts.

Major allergens Der p 1, Der p 2, Der p 23, Der f 1, Der f 2, Der f 23, Blo t 5, Blo t 2 and Blo t 21 were identified in the corresponding native, depigmented, as well as in depigmented-polymerized extracts.

The presence of all major allergens in native, depigmented and depigmented-polymerized extracts of D. pteronyssinus, D. farinae and B. tropicalis has been confirmed. In addition, most secondary allergens were also identified in the three types of extracts.