Figure 1.
Purification of allergens from the wasp venoms (WV), V. magnifica.
A: WV aliquot of 0.2 g was applied to a Sephadex G-75 gel filtration. The absorbance of the eluate was monitored at 280 nm. B: Fraction 1 from Sephadex G-75 gel filtration was subjected to AKTA Resource S cationic exchange chromatography. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. C: The first protein peak after Resource S cationic exchange chromatography (peak 1.1) was subjected to AKTA Mono S cationic exchange chromatography. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. D: The fifth protein peak after Resource S cationic exchange chromatography (peak 1.5) was subjected to AKTA Mono S cationic exchange chromatography. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. E & F: SDS-PAGE analysis of purified allergens of Vesp ma 5 and Vesp ma 2 in 15% gel concentration. R: reduced; NR: non-reduced.
Figure 2.
cDNA encoding the Vesp ma 5 (A) and Vesp ma 2 (B) from the wasp venom of V. magnifica and deduced amino acid sequence.
The amino acid sequences of peptide fragments determined by Edman degradation are underlined. The predicted signal peptide is in italic. -: stop codon.
Figure 3.
Immunoblot of IgE reactivity to Vesp ma 5(A), Vesp ma 2(B), Tab y 5(C), and Tab y 2(D).
Lanes 1–8, representatives of subject serum from patients with wasp allergy; Lanes I–VIII, representatives of subject serum from patients with horsefly allergy; Lanes 9 and 10, negative controls. E: Three allergens (Vesp ma 5, Vesp ma 2, Tab y 2) were mixed and loaded on the same lane of SDS-PAGE (Left), and transferred to a nitrocellulose membrane for immunoblotting (Right). Comassie blue staining was performed to visualize proteins after PAGE-SDS. Note: Tab y 5 was not mixed with Vesp ma 5, Vesp ma 2, and Tab y 2 because it has the same molecular weight with Vesp ma 5.
Figure 4.
Cross ELISA inhibition of purified wasp and horsefly allergens with patients' serum from subject 2, 3 and 8.
A& B: 20 µg/ml coated wasp venom's binding with serum IgE from subject 2 (A) and 8 (B) was inhibited by wasp venom, horsefly SGE, Vesp ma 5, Vesp ma 2, Tab y 5, and Tab y 2, respectively. C& D: 20 µg/ml coated horsefly SGE's binding with serum IgE from subject 3 (C) and 8 (D) was inhibited by wasp venom, horsefly SGE, Vesp ma 5, Vesp ma 2, Tab y 5, and Tab y 2, respectively. Inhibitor concentrations ranged from 10−5 µg to 20 µg/ml. SGE: salivary gland extracts; V-antigen: Vesp ma 5; V-hya: Vesp ma 2; S-antigen: Tab y 5; S-hya: Tab y 2.