Mapping of Dermatophagoides farinae mite allergens by two-dimensional immunoblotting.

Abstract

Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized. This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis. D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. 2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der 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 peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus. 2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.