14 Comprehensive Expression of Recombinant House Dust Mite Allergens for Component-Resolved Diagnosis

Pooideae species. While for several species the most important allergens (group 1 and group 5) have been identified, other allergens have either not been identified or sequence data are still missing. We have used mass spectrometry (MS) together with genetic and immunological methods to identify allergens in various grass pollen extracts. Methods: Pollen extracts of 6 different grass species (Phleum pratense, Holcus lanatus, Lolium perenne, Dactylus glomerata, Festuca pratensis, Poa pratensis) and a mixture thereof were analyzed. For identification of allergens by MS, extracts were subjected to enzymatic digestion. Resulting peptides were separated by liquid chromatography and analyzed by tandem mass spectrometry. Protein identification was performed by searching both the NCBIPlant release and an individually designed database. The presence of individual allergens was confirmed with allergenspecific monoclonal antibodies. Unknown sequences were determined following cDNA synthesis from pollen RNA and allergen sequence amplification by PCR. Results: Fes p 1 and Fes p 5 were identified by the PCR approach. MS analysis of pollen extracts from the 6 individual species resulted in detection of all known allergens including the newly identified Fes p 1 and Fes p 5. Based on the homology of allergens from different grass species, previously unknown sequences of representatives of groups 2, 3, 4, 7, 11, 12 and 13 were detected by MS in investigated extracts with high sequence coverage. Group 6 allergens could not be identified in some of the analyzed extracts. These findings are supported by immunological analyses and thus demonstrate the specificity of the applied method. Members of all allergen groups were identified in an extract mix prepared from pollen of all 6 grass species studied. Conclusions: The most important grass allergens (group 1 and group 5) were detected in all extracts. In addition all other known allergens of the assayed species and homologues thereof could also be identified, thus demonstrating the quality of the tested extracts.