In France and Finland, farmer's lung disease (FLD), a hypersensitivity pneumonitis common in agricultural areas, is mainly caused by Eurotium species. The presence of antibodies in patients' serum is an important criterion for diagnosis. Our study aimed to improve the serological diagnosis of FLD by using common fungal particles that pollute the farm environment as antigens. Fungal particles of the Eurotium species were observed in handled hay. A strain of Eurotium amstelodami was grown in vitro using selected culture media; and antigen extracts from sexual (ascospores), asexual (conidia), and vegetative (hyphae) forms were made. Antigens were tested by enzyme-linked immunosorbent assay (ELISA), which was used to test for immunoglobulin G antibodies from the sera of 17 FLD patients, 40 healthy exposed farmers, and 20 nonexposed controls. The antigens were compared by receiver operating characteristic analysis, and a threshold was then established. The ascospores contained in asci enclosed within cleistothecia were present in 38% of the hay blades observed; conidial heads of aspergillus were less prevalent. The same protocol was followed to make the three antigen extracts. A comparison of the results for FLD patients and exposed controls showed the area under the curve to be 0.850 for the ascospore antigen, 0.731 for the conidia, and 0.690 for the hyphae. The cutoffs that we determined, with the standard deviation for measures being taken into account, showed 67% for sensitivity and 92% for specificity with the ascospore antigen. In conclusion, the serological diagnosis of FLD by ELISA was improved by the adjunction of ascospore antigen.Eurotium amstelodami is known to cause hypersensitivity pneumonitis (HP), of which farmer's lung disease (FLD) is a common form (24,29). Eurotium species, widely isolated from hay harvested in several European countries (22, 30), constitute a major etiological agent of the disease in France and Finland (13,26). Thus far, the diagnosis of HP has most often relied on an array of unspecific clinical symptoms and signs developed in an appropriate setting and the demonstration of interstitial markings on chest radiographs, serum antibodies against offending antigens, a lymphocytic alveolitis on bronchoalveolar lavage (BAL), and the granulomatous reaction on lung biopsy specimens (17, 21). Enzyme-linked immunosorbent assay (ELISA) is one of the techniques that can be used to look for specific serum antibodies (6). Although ELISA indicates significantly higher immunoglobulin G antibody levels in patients with FLD than in control groups, no clear threshold has been established due to an extreme variability in the results that have been obtained (6,25). The antigens used in most studies derive from the sonication of crude fungi.E. amstelodami can be found in any one of three forms (sexual, asexual, or vegetative), and the one that is predominant in the agricultural environment is not known to date. Sexual reproduction is characterized by the production of cleistothecia containing asci wit...