Molecular Reference Materials for Standardization of Allergen Measurements

Abstract

Allergen measurements are widely used for determination of the potency of allergy vaccines, for environmental exposure assessments, and for validation of IgE molecular diagnostics. With the exception of Bet v 1 and Phl p 5a, few purified allergen standards have been developed. The aim of this study was to produce purified allergen standards, with defined specifications, that could serve as Molecular Reference Materials (MRM) for use in allergy diagnostics, enzyme- or fluorescent immunoassays, and multiplex arrays. Natural and recombinant food, dust mite, pollen and animal allergens (n = 11) were purified by affinity and/or gel-filtration chromatography and analyzed by SDS-PAGE, LC-MS/MS, ELISA, and FEIA or chimeric IgE ELISA. Natural allergens from peanut, dust mite and animal were lyophilized using different buffer conditions. Real time stability data are collected from frozen liquid allergens and lyophilized allergens. Allergen purity, assessed by LC-MS/MS after Trypsin digest and /or silver-stained SDS-PAGE, was >95%. Real time stability tests of frozen liquid allergens (up to 24 months) and frozen lyophilized allergens, showed comparable potency in allergen-specific ELISA and no signs of degradation on SDS-PAGE. Monoclonal antibody ELISA and IgE reactivity of recombinant allergens showed excellent correlations with natural allergens. Optimized, ISO 9001 compliant, bioprocessing pathways have been established to yield pure, immune-reactive natural and recombinant allergens. These MRM are stable and will improve the standardization of allergy diagnostics and vaccines used for immunotherapy.