IDENTIFICATION OF THE REACTIVE SUBUNITS OF ASPERGILLUS-UMBROSUS INVOLVED IN THE ANTIGENIC RESPONSE IN FARMERS LUNG

Background Farmer's lung (FL) is the most common form of extrinsic all ergic alveolitis and the fungus Aspergillus umbrosus has frequently be en associated with FL among Finnish farmers. IgG and IgA class antibod ies against Aspergillus umbrosus are found in the serum of FL patients and healthy exposed farmers. So far the immunologically reactive subu nits of Aspergillus umbrosus are unknown. Objectives The purpose of th e study was to identify and localize the reactive subunits of Aspergil lus umbrosus that are involved in the antigenic response in FL. Method s The distribution of the primary antibody on the outer wall layers of 4- and 11 day-old fungi Aspergillus umbrosus was studied by pre-embed ding immunolabelling with fluorescein isothiocyanate (FITC)- and gold- conjugated antibodies. The surface carbohydrates of the outer wall lay ers were studied using FITC-conjugated lectins. Results FITC-conjugate d antibodies from FL patient sera reacted with spores (conidia), phial ides and hyphae, whereas the fruiting body, cleistothecia, remained un stainable. Immunofluorescence labelling was confirmed by immunolabelli ng 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 particl es were found scattered on the surfaces of cleistothecia. The surface carbohydrates of Aspergillus umbrosus conidia, vesicles, phialides and hyphae had a high affinity to GlcNAc beta 1- and Manc alpha 1-/Glc al pha 1- specific lectins and only GlcNAc beta 1-specific lectins also r eacted with the outer cell walls of cleistothecia. Blocking of the ant igenic sites of Aspergillus umbrosus by preincubating antigen-coated e nzyme linked immunosorbent assay (ELISA) plates with lectins caused so me inhibition, indicating that sites with the highest affinity fdr lec tins were not always antigenic sites. Conclusion Gold- and FITC-conjug ated antibodies were distributed mostly on the outer wall layers of co nidia, hyphae and phialides of Aspergillus umbrosus and GlcNAc beta 1- linked lectins had the strongest affinity to the outer wall layers.