RATIONALE: Paecilomyces variotii is a hydrophilic fungal species. Preliminary studies have identified P. variotii as an aeroallergen, occupational hazard, and potential biomarker of indoor fungal exposure. In this study, we characterized the allergens derived from P. variotii. METHODS: A diverse collection of participant sera that were positive to a fungal skin prick test or ImmunoCAP (n524) were selected for analysis. Hyphal extracts derived from P. variotii were separated by SDS-PAGE or 2D gel electrophoresis and immunostained with patient serumIgE following western blotting. Immunostained P. variotii antigens were excised and sequenced using nanoscale Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (nanoscale UPLC-MS/MS). De-identified sera from 6 healthcare workers (HCW) and 2 non-atopics were used as controls. RESULTS: Immunoblotting of P. variotii hyphal extracts revealed IgE against 25 antigens within the range 18-132 kDa. IgE reactivity towards 25, 29, 44, and 65 kDa antigens bound IgE from 88%, 96%, 88%, and 58% of study participants, respectively. These immundominant allergens were identified by nanoscale UPLC-MS/MS to be 60S ribosomal protein L10 (25 kDa), electron transfer flavoprotein B subunit (29 kDa), ketol acid reductoisomerase (44 kDa), and heat shock protein 70 (65 kDa). Other antigens that bound IgE did not return significant matches from the protein databank. No IgE antibodies were identified in non-atopic controls. CONCLUSIONS: Participants with specific IgE to P. variotii reacted most commonly with higher molecular weight (MW) antigens. Four immunodominant allergens were identified to bind IgE in greater than 50% of the study participants.
Fungi; Allergens; Biomarkers; Indoor-air-pollution; Indoor-environmental-quality; Sensitivity-testing; Skin-tests; Immune-reaction; Immunodiagnosis; Immunological-tests; Antigens; Nanotechnology; Liquid-chromatography; Mass-spectrometry; Health-care-personnel; Proteins; Antibody-response; Molecular-biology; Molecular-structure