The stability of house dust mite allergens in glycerinated extracts.
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.
Soldatova
L N
LN
Laboratory of Immunobiochemistry, Division of Allergenic Products and Parasitology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, USA.
Paupore
E J
EJ
Burk
S H
SH
Pastor
R W
RW
Slater
J E
JE
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
D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D011480:Protease Inhibitors; D014020:Tissue Extracts; D005990:Glycerol