397 Standardization and Characterization of Dust Mite Extracts Manufactured in the USA

BACKGROUND: Standardized Dermatophagoides dust mite extracts are produced in the US from purified whole bodies. Growth media and the processes for separating mites from media vary among manufacturers. The FDA requires that mite extracts are standardized and labeled in AU/mL. Potency is determined using a laboratory ELISA competition method to compare the product with an FDA reference. The method measures binding of IgE from an FDA supplied sera pool to antigens bound to an ELISA plate and AU is calculated from the ability of the test extract to inhibit the binding relative to the 10,000 AU/mL FDA reference. Since this is the only FDA requirement for potency, the purpose of this study was to compare mite extracts from different US manufacturers for protein complexity, major allergen, and potency using various biochemical characterization techniques. METHODS: Der group 1 and 2 allergens were measured in mite extracts from several manufacturers produced over the last 8 years using validated ALK immunoassays. Competition IgE binding was performed using FDA references and sera pools. The effect of the immobilized extract on the relative potency compared to the FDA reference was determined. Protein profiles were determined using SDS-PAGE. RESULTS: The average Der 1 and Der 2 levels and ratio in 10,000 AU/mL products varied considerably (Der 1: 25–140 μg/mL, Der 2: 2–140 μg/mL). The ratio of Der 1/Der 2 was manufacturer related and ranged from 1:1 to more than 10:1. The extract used to coat the ELISA plates had a marked impact on Relative Potency (RP) with up to a 3-fold difference. RP determined by competition IgE binding was correlated with major allergen content but the difference in potency was obtained by coating with different batches of 10,000 AU/mL mites. CONCLUSIONS: Often called a “total” IgE test, the competition IgE ELISA is highly dependent on the allergen used to coat the plastic microplate. US mite extracts with the same AU/mL can have very different Der 1 and 2 content.