Isolation and characterization of the 68 kD allergen from house dust mite Dermatophagoides pteronyssinus

Abstract

House dust mites (HDM) represent a major source of allergens, contributing to the increasing incidence of type I hypersensitivity disease worldwide. Over 30 different IgE-binding proteins from the HDM extract were detected. Although group 1 and 2 have been identified as major allergens, due to the safety and efficacy of allergy diagnosis and immunotherapy, there is a need to carefully evaluate the clinical relevance of other allergens present in the HDM extract. In regard to this, a high molecular mass allergen of about 68 kD was purified from the HDM extract using a combination of gel permeation chromatography and reversed-phase chromatography. The IgG and IgE reactivity of the purified protein were preserved during the purification process, as confirmed by Western blot analysis with polyclonal rabbit antibodies and dot blot analysis with a pool of sera from subjects with house dust mite allergy, respectively. In addition, the IgE reactivity was confirmed using ELISA testing with nine patient sera. The biological potency of the 68 kD allergen was confirmed by skin prick testing in five allergic subjects, suggesting that the high molecular mass allergen is a good candidate for component-resolved diagnosis of house dust mite allergy and eventual therapeutic treatment.

Grinje iz kućne prašine predstavljaju jedan od glavnih izvora alergena koji su u značajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujućih proteina iz kućne prašine je detektovano do danas. Alergeni grupe 1 i 2 označeni su kao glavni alergeni kućne prašine. Međutim, da bi se poboljšala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kućne prašine, neophodno je odrediti klinički značaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kućne prašine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost prečišćenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zečijim antitelima na ekstrakt kućne prašine i 'pool'-om seruma osoba alergičnih na kućnu prašinu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na prečišćeni protein u ELISA testu. Biološka reaktivnost prečišćenog alergena je potvrđena u kožnim probama na pet pacijenata, ukazujući da je prečišćen alergen dobar kandidat za dijagnozu alergije na kućnu prašinu pojedinačnim komponentama i eventualni terapeutski tretman.