The epitope stability of group 1 and group 2 allergens in mite extracts.

BACKGROUND

Commercial mite allergenic extracts sold in the US are prepared with whole body mites in buffer solutions containing 50% glycerol. Mite extracts were reported to contain large number of proteolytic enzymes and their structural integrity in aqueous solutions have not been evaluated.

OBJECTIVE

To evaluate the epitope stabilities of Group 1 and 2 allergens in two commercial mite extracts used by Center for Biologics Evaluation and Research (CBER) as reference extracts, E5-Dp (Dermatophagoides pteronyssinus) and E5-Df (D. farinae).

METHODS

Epitope stability was determined by using monoclonal antibodies in a Sandwich ELISA. Samples were stored at four different temperatures and the amounts of Der p 1, Der p 2, Der f 1, and Der f 2 were determined at different time intervals. The overall stability of mite extracts was evaluated by immunoblot and competition ELISA.

RESULTS

The epitope stability of these allergens varies: Der f 1 was stable for at least 3 years and Der f 2 for 1 year when stored at 4 degrees C; Der p 1 and 2 were less stable. None of the Group 1 and 2 allergens remained intact when stored at 50 degrees C. Immunoblot and competition ELISA data also showed similar trend of degradation as compared with extracts stored at 4 degrees C for same length of time.

CONCLUSION

With the exception of Der f 1, the amount of detectable epitopes in Group 1 and 2 allergens reduce rapidly after 1 year, especially at elevated temperatures. The changes in allergen composition were also observed by immunoblotting and in relative potency by ELISA competition assay. These findings are highly relavent to the users of CBER's mite extracts as standards.