MAPPING OF DERMATOPHAGOIDES-FARINAE MITE ALLERGENS BY 2-DIMENSIONAL IMMUNOBLOTTING

Background: Allergens from the house dust mite Dermatophagoides farina e are responsible for frequent respiratory allergic disorders, but onl y 3 groups of these allergens are well characterized. Objective: This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis, Methods: D farinae mite a llergens, extracted from whole cultures in the presence of a mild dete rgent, were separated by 2-D electrophoresis with subsequent immunoblo tting. IgE-binding proteins were detected with individual mite-sensiti ve patient sera and the anti-D pteronyssinus human serum pool. Allerge ns were identified by an inhibition immunoblot test, by means of speci fic mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. Results: 2-D immunoblotting with ind ividual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-b inding patterns. In addition to identification of allergens Der f 1, D er f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of pept ides from the latter allergens revealed significant homologies with al lergen Mag 3 from D farinae and with a chitinase from prawn Penaeus ja ponicus. Conclusion: 2-D electrophoresis with subsequent immunoblottin g and protein microsequencing allowed characterization of a more compl ete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.