Caracterización del Cultivo del Ácaro del Polvo D. Siboney como Materia Prima para la Produccion de Extractos Alergénicos.

Dermatophagoides siboney is House Dust Mite specie, described and reported for the first time in Cuba in 1982, where it has a high incidence on the allergic population. We have developed and maintained a massive culture of this mite in laboratory conditions for 10 years, having fit the optimal growth parameters related to temperature and relative humidity. With the objective to improve the standardization of the allergen vaccines used for diagnosis and immunotherapy, we have considered to perform a characterization of the culture, regarding the allergenic potency, mRNA content, enzymatic activity and content of Der s 1 major allergen. Mites were grown in a hypoallergenic diet during 10 weeks. Inhibition ELISA was performed to determine the biological activity, using serum of allergic patients. The Der s 1 content was assessed using monoclonal antibodies ELISA. The presence of the Der s 2 mRNAs was detected by means of PCR using specific primers. The identity of both proteins was demonstrated by SDS-PAGE and Western-blotting IgE. For the determination of the cysteine-protease enzymatic activity of the Der s 1 allergen, a kinetic test was carried out using casein as substrate. The allergenic potency reached values of more than 10 000 Biological Units per ml. The content of Der s 1 varied between 10- 350 µg/ml. The selected oligo-nucleotides allowed to amplify a fragment of cDNA of 391 pairs of bases, that codifies for Der s 2 mature protein. Amplification of Der s 2 cDNA was noted already even in the latency phase of the culture. The extracts obtained from cultures in the exponential phase showed greater allergen potency and mRNA Der s 2 content. The extract showed cysteine-protease activity, which increased after 6 weeks of culture. The greatest Der s 1 content was observed during the last weeks of the culture, which corresponds with the fecal origin of this protein. [ABSTRACT FROM AUTHOR]