9 A Proteomic Style Approach to Characterize a Grass Mix Product Reveals Potential Immunotherapeutic Benefit

BACKGROUND: Grass allergy immunotherapies often consist of a mix of different grass extracts each containing several proteins of different physiochemical properties; however the subtle contributions of each protein are difficult to elucidate. This study aimed to identify and characterise the group 1 and 5 allergens in a 13 grass extract and to standardise the extraction method. METHODS: The grass pollens were extracted in isolation and pooled and also in combination and analysed using a variety of techniques including enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC-MS) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Gold-staining and IgE immunoblotting revealed a high degree of homology of protein bands between the 13 species and the presence of a densely stained doublet at 25 to 35 kD along with protein bands at approximately 12.5, 17 and 50 kD. The doublet from each grass species demonstrated a high level of group 1 and 5 interspecies homology. However, there were a number of bands unique to specific grasses consistent with evolutionary change and indicative that a grass mix immunotherapeutic could be considered broad spectrum. CONCLUSIONS: SDS-PAGE and IgE immunoblotting showed all 13 grasses share a high degree of homology particularly in terms of group 1 and 5 allergens. IgE and IgG ELISA potencies were shown to be independent of extraction method.