title: The stability of house dust mite allergens in glycerinated extracts.
abstract: BACKGROUND
  Mite allergen vaccines are important diagnostic and immunotherapeutic reagents. Previous studies on mite allergen stability under different storage conditions have yielded contradictory results.
  
  
  OBJECTIVE
  We sought to compare, over a 12-month period, the stability of mite allergens reconstituted in 50% glycerol and stored at different temperatures 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 degrees 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 immunoblot analyses.
  
  
  RESULTS
  The overall potencies of the stored extracts measured by competition ELISA were stable at -20 degrees C and 4 degrees C. As determined by means of the immunoblot and 2-site ELISA, Der f 1 levels decreased at 4 degrees C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of the allergen-specific assays. Storage at 37 degrees C led to overall loss of potency and allergen content, whereas storage at -70 degrees C was associated with a moderate loss of potency that increased with multiple freeze-thaw cycles. Protease inhibitors had no effect on allergen stability.
  
  
  CONCLUSIONS
  Although overall potency of the extracts, as measured by competition ELISA, was preserved at -20 degrees C and 4 degrees C, allergen-specific assays indicated loss of allergens. These findings suggest that the competition ELISA is insensitive to decreases in the concentrations of individual allergens.
authors.affiliation: Laboratory of Immunobiochemistry, Division of Allergenic Products and Parasitology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, USA.
authors.forename: L N
authors.initials: LN
authors.lastname: Soldatova
authors.forename: E J
authors.initials: EJ
authors.lastname: Paupore
authors.forename: S H
authors.initials: SH
authors.lastname: Burk
authors.forename: R W
authors.initials: RW
authors.lastname: Pastor
authors.forename: J E
authors.initials: JE
authors.lastname: Slater
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D039741:Antigens, Dermatophagoides; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005990:Glycerol / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry* / Q000187:drug effects / Q000592:standards; D015151:Immunoblotting; D008925:Mites / Q000276:immunology; D011480:Protease Inhibitors / Q000494:pharmacology; D014020:Tissue Extracts / Q000276:immunology* / Q000592:standards
chemical_list: D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D011480:Protease Inhibitors; D014020:Tissue Extracts; D005990:Glycerol