Household Dust Mite Allergens In Experimental Research

House dust mites (HDM) is one of the most important indoor allergens. Substantial evidence shows a significant increase in the world-wide prevalence of allergy as well as allergic disorders to HDM during the last decades. Allergy to HDM can cause allergic rhinititis (AR), allergic asthma and allergic skin disorders. Allergic reaction to HDM can occur in every age stage of the allergic people, also HDM is the most common allergen in allergic asthma in children. Accordingly, HDM has been one of the focuses on allergeology international.Allergen Preparation (Extraction):Raw material of Dermatophagoides pteronyssinus (D. pteronyssinus) purified mite bodies (PMB) was purchased from Allergon Inc. (Sweden). The extracts were prepared with Coca's solution, lysis solution of two-dimensional (2D) electrophoresis and Trizol reagent respectively. The protein concentrations in the extracts by different methods were: C₀: C₁: L: T=5.7: 6.3: 9: 8; Their sequence was ranked as follows: lysis solution >Trizol reagent >Coca's solution. In protein concentration assay, BCA reagents were used with good results.Two-dimensional electrophoresis :2D electrophoresis was done with different extracts from D. pteronyssinus PMB as stated above. The experiment results were : 1) many protein spots at low molecular weight(MW) in extract with Coca's solution method; 2) more protein spots at low MW in extract with lysis solution method; 3) several medium MW protein spots(e. g. 174-178Kd ) and more low MW protein spots in extract with Trizol reagent. It was showed that Trizol reagent extracted more protein components of D. pteronyssinus PMB than that by Coca's solution and lysis solution.Six peculiar protein spots were identified in 2D electrophoresis map of D. pteronyssinus PMB extract with Trizol reagent using Coomassie blue stain. They were two isolated acid proteins and four proteins closing in neutral pH range. One spot of acid proteins showed pI and MW as 4.4 and 133Kd, the another was 4.81 and 35.4Kd respectively. Two protein spots from four neutral proteins showed pI as 7.2, and MW as 49.5Kd and 46.4Kd respectively, The remaining two protein spots showed pI as 6.9, and MW as 50.2Kd and 47.0Kd respectively. These six peculiar proteins were present in every 2D map in our experiment (twelve's times). It might be considered as one of finger printing of D. pteronyssinus PMB in 2D electrophoresis map.Two-dimensional immunoblotting :Two-dimensional immunoblotting (2D immunoblotting) were carried out after 2D electrophoresis of D. pteronyssinus PMB extract with Trizol reagent.Identification of IgE-binding components of D. pteronyssinus (Der p ) PMB with MS pool were as follows:...