Figure 1.
Allergen purification from DME.
A: 380-75 (Superfine, Amersham Biosciences; 26×100 cm), the absorbance of eluted fractions were monitor at 280 nm. B: Pooled fraction I was dialyzed against 20 mM Tris-HCl, pH 8.0 and subjected to further purification by Resource Q anionic exchange column equilibrated with 0.02 M Tris-HCl, pH 8.0 on an AKTA system, and eluted at a flow rate of 1 ml/min with the indicated NaCl gradient in 0.02 M Tris-HCl pH 8.0. Inserts: Purified proteins were subjected to SDS-PAGE analysis on a 15% gel. R, reduced; UR, unreduced.
Figure 2.
The identification of D. farinae allergens with molecular weight around 90 kDa by coupling 2-DE with 2-D immunoblotting.
Fraction I from Sephadex G-75 gel filtration was further separated by 2-DE and stained with Coomassie G-250 (A) or transferred to PVDF membranes followed by IgE immunoblotting with dust mite allergic (B) and healthy human sera (C), respectively.
Figure 3.
Amino acid sequences of seven interior fragments (A–G) determined by ESI-QUAD-TOF mass spectrometry analysis.
Figure 4.
cDNA sequence encoding Der f 24 and the deduced amino acid sequence.
The sequences underlined were determined by mass spectrometry analysis and Edman degradation. *: stop codon. Bolded sequences are calponin homology domains (actin-binding domains); Shaded seqeunces are spectrin domains; Bolded and italized squences are EF-hand motifs.
Figure 5.
Specific IgE reactivity to allergen Der f 24.
A: Immunobloting analysis of specific IgE reactivity to allergen Der f 24 in the sera from the patients with dust mite allergy. Lanes 1–9, representatives of sera from allergic subjects. Lane 10 & 11 marked as C, sera from healthy individuals as negative control. B: Evaluation of specific IgE reactivity to allergen Der f 24 by direct ELISA. Group 1: the sera from healthy control subjects, group 2: the sera from Der f 24-positive patients.
Figure 6.
Purified allergens inhibited the patients’ serum IgE binding to the coated DME in a dose-dependent manner (A, B) and induction of basophil activation by purified Der f 24 (C, D).
A and B represent the serum from dust mite allergy patients 6 and 8, respectively. Allergen concentrations ranged from 0 to 30 µg/ml. DME: dust mite extracts. Der f 24 was incubated with 6 dust mite allergenic patients’ PBMC (C) or 6 healthy subjects’ PBMC (D). Group 1: using the stimulation buffer to evaluate the basal CD63 level. Group 2: Der f 24.
Table 1.
Results of skin prick tests.