The stability of house dust mite allergens in glycerinated extracts

Background: Mite allergen vaccines are important diagnostic and immunothera peutic reagents. Previous studies on mite allergen stability under differen t storage conditions have yielded contradictory results, Objective: We sought to compare, over a 12-month period, the stability of m ite allergens reconstituted in 50% glycerol and stored at different tempera tures and to examine the role of protease inhibitors in enhancing allergen stability. Methods: Lyophilized allergen extracts were reconstituted in 50% glycerol, with and without protease inhibitors, and stored at -70 degrees C, -20 degr ees C, 4 degrees C, or 37 degrees C for 12 months. At 6 and 12 months, the extracts were compared with freshly dissolved extracts by competition ELISA with pooled allergic sera, 2-site ELISA with mite-specific mAbs, and immun oblot analyses, Results: The overall potencies of the stored extracts measured by competiti on ELISA were stable at -20 degrees C and 4 degrees C. As determined by mea ns of the immunoblot and 2-site ELISA, Der fl levels decreased at 4 degrees C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of th e allergen-specific assays. Storage at 37 degrees C led to overall loss of potency and allergen content, whereas storage at -70 degrees C was associat ed with a moderate loss of potency that increased with multiple freeze-thaw cycles, Protease inhibitors had no effect on allergen stability. Conclusion: Although overall potency of the extracts, as measured by compet ition ELISA, was preserved at -20 degrees C and 4 degrees C, allergen-speci fic assays indicated loss of allergens. These findings suggest that the com petition ELISA is insensitive to decreases in the concentrations of individ ual allergens.