METHODS: To develop a new and improved multiplex assay using a dynamic combination of high throughput LC/MS technology, multiple reaction monitoring (MRM) MS, and immunoassay for an in-depth allergenic protein identification, and simultaneous quantification, potency and stability measurements.
RESULTS: The method development process involves immunoallergomics to establish a comprehensive profile of IgE-reactive proteins in extracts, and generate a peptide library of all allergens, isoforms, and variants. Prototypic and quantotypic peptides were selected for MRM method development for accurate quantification of all GCr allergens. ICH (Q2(R1)) guidelines were followed to assure assay robustness and suitability. Allergens from various commercial GCr extracts were quantified to show considerable variability between preparations. The LC-MRM MS method was also evaluated for as a stability assay of allergens subjected to various degradative conditions (temperature, pH, and enzymes). The MRM-based potency of selected allergens correlates well with ELISA-based measurements. Also, the LC-MRM was evaluated for its application towards measurement of GCr allergens in dust samples collected from various US households.
CONCLUSIONS: The new assay is robust and valuable for potency measurement of GCr allergen extract and offers great promise in achieving full characterization.