IgE immunoblot reactivity in allergic fungal sinusitis

Abstract

P. S. Hutcheson1, M. S. Schubert2, R. G. Slavin1; 1Internal Medicine/Allergy & Immunology, Saint Louis University School of Medicine, St. Louis, MO, 2Allergy Asthma Clinic, Ltd., Phoenix, AZ. RATIONALE: Although patients with allergic fungal sinusitis (AFS) have IgE skin reactivity to the etiologic fungus, the role of IgE in the disease has been questioned. Since the identification of the etiologic fungus is sometimes difficult and serial cultures from some AFS patients may grow several different fungi, we attempted to identify possible crossreacting fungal antigens. METHODS: Ten patients with Bipolaris spicifera AFS were enrolled and IRB-approved consent obtained. IgE immunoblots utilized 25ug protein from 7 fungal extracts: Alternaria alternata, Aspergillus fumigatus, Bipolaris sorokiniana, Bipolaris/Curvularia spicifera, Epicoccum purpurascens, Fusarium moniliforme, and Phoma betae. Nitrocellulose membranes were incubated with patient serum, goat anti-human IgE, bovine alkaline phosphatase conjugated anti-goat IgG, and BCIP/NBT chromogen. RESULTS: Nine out of ten Bipolaris spicifera AFS patients exhibited IgE to all 7 fungi tested. One patient lacked IgE to Aspergillus and Phoma. Positive IgE bands were seen from ~10 kD-160 kD, with Bipolaris/Curvularia producing the most positive bands. No single molecular weight band appeared among the 7 fungi, and not all patients reacted to the same molecular weight antigens. CONCLUSION: All of our Bipolaris spicifera AFS patients showed some degree of IgE cross reactivity to 6 other fungi, although no single molecular weight antigen across all species was seen. The most intense bands were produced from the AFS-causative mold Bipolaris/Curvularia spicifera. A relationship between overall degree of fungal IgE immunogenicity and clinical disease is suggested. Funding: Saint Louis University School of Medicine 214 Study on Dermatophagoides pteronyssinus Allergens Fingerprinting by Two-Dimensional Immunoblotting J. Sun1, H. Zhang1, W. Ying2, X. Qian2; 1Dept. of Allergy, Peking Union Medical College Hospital, Beijing, CHINA, 2Center of Proteiomics, Beijing Radiation Institute, Beijing, CHINA. RATIONALE: To explore Dermatophagoides pteronyssinus (D. pteronyssinus) Allergens finger printing with two-dimensional (2D) electrophoresis and 2D immunoblotting. METHODS: D. pteronyssinus purified mite bodies (PMB) extracts were prepared with Trizol reagent. In protein concentration assay, BCA reagents were used. Mixed sera pool consisted of twelve D. pteronyssinus allergic patients. 2D electrophoresis and 2D immunoblotting were done with D. pteronyssinus. extract and mixed sera pool. RESULTS: Identification of IgE-binding components of D. pteronyssinus (Der p)PMB with MS pool were as follows: Der p 1(pI 5.4, Mr=25.6Kd);Der p 2(pI 7.6-9.0, Mr=15.2Kd);Der p 3(pI 8.1-8.5, Mr=28.5-28.6Kd);Der p 4(pI 6.5-7.0, Mr=61.0-62.0Kd);Der p 5(pI 7.0-7.3, Mr=14.8Kd);Der p 6(pI 5.86.1, Mr=25.5-25.8Kd);Der p 7(pI 6.9-7.1, Mr=26.5Kd);Der p 8(pI 6.8-7.0, Mr=24.9Kd);Der p 9(pI 8.8, Mr=28.1Kd);Der p 10(pI 6.5-6.8, Mr=36.4Kd). Some allergens similar to Le Mao described: C(pI 8.4-8.9, Mr=20Kd);D(pI 7.4-8.5, Mr=37.6-38.6Kd),E(pI 7.0-7.6, Mr=40.5-41.5Kd); F(pI 5.3, Mr=43.8Kd).Der p 14 (pI 6.5-6.9,175-180Kd) was initially showed in 2D immunoblotting in this study. Der p 11 (pI 6.9-7.3, Mr=90-93Kd). Der p 16 (pI 6.98-7.38, Mr=57.0-56.0Kd);Der p 18(pI 7.1-7.5, Mr=69.7-70.7Kd);isoform of Der p 5(9.0, Mr=14.8Kd). CONCLUSIONS: D. pteronyssinus allergens in 2D finger printing could be seen their pI and Mr at the same map. In addition, Der p 14 and other new components immunoblotted showed that more components immunoblotted in D. pteronyssinus could be found if we extract it with Trizol reagent.