Identification of the reactive subunits of Aspergillus umbrosus involved in the antigenic response in farmer's lung

Background Farmer's lung (FL) is the most common form of extrinsic allergic alveolitis and the fungus Aspergillus umbrosus has frequently been associated with FL among Finnish farmers. IgG and IgA class antibodies against Aspergillus umbrosus are found in the serum of FL patients and healthy exposed farmers. So far the immunologically reactive subunits of Aspergillus umbrosus are unknown. Objectives The purpose of the study was to identify and localize the reactive subunits of Aspergillus umbrosus that are involved in the antigenic response in FL. Methods The distribution of the primary antibody on the outer wall layers of 4- and 11 day-old fungi Aspergillus umbrosus was studied by pre-embedding immunolabelling with fluorescein isothiocyanate (FITC)- and gold-conjugated antibodies. The surface carbohydrates of the outer wall layers were studied using FITC-conjugated lectins. Results FITC-conjugated antibodies from FL patient sera reacted with spores (conidia), phialides and hyphae, whereas the fruiting body, cleistothecia, remained unstainable. Immunofluorescence labelling was confirmed by immunolabelling electron microscopy, and gold-conjugated IgG antibodies were found on the outer cell walls of spinulose conidia, and in a less degree on the outer cell walls of hyphae and phialides. Only single gold particles were found scattered on the surfaces of cleistothecia. The surface carbohydrates of Aspergillus umbrosus conidia. vesicles, phialides and hyphae had a high affinity to GlcNAcβl- and Manαl-/Glcαl- specific lectins and only GlcNAcβl-specific lectins also reacted with the outer cell walls of cleistothecia. Blocking of the antigenic sites of Aspergillus umbrosus by preincubating antigen-coated enzyme linked immunosorbent assay (FLISA) plates with lectins caused some inhibition, indicating that sites with the highest affinity for lectins were not always antigenic sites. Conclusion Gold- and FITC-conjugated antibodies were distributed mostly on the outer wall layers of conidia, hyphae and phialides of Aspergillus umbrosus and GlcN Acβl -linked lectins had the strongest affinity to the outer wall layers.