Purification of the antigenic components of pigeon dropping extract, the responsible agent for cellular immunity in pigeon breeder's disease

Background: Pigeon breeder's disease (PBD) is a lung disease caused by inha lation of antigens derived from pigeons. Objective: This study was undertaken to characterize the responsible compon ent of pigeon dropping extract (PDE) for PBD. Methods: First, crude PDE was applied to SDS-PAGE followed by immunoblottin g by using antibodies in bronchoalveolar lavage (BAL) fluid. Second, 9 band s of PDE were separated by SDS-PAGE and used for antigen-induced PBMCs. Fin ally, amino-terminal sequencing was conducted on an isolated 21-kd protein by 2-dimensional electrophoresis. Results: Immunoblots with BAL fluid from patients with PBD identified 9 ban ds. Similar patterns were observed by using BAL fluid from 10 control patie nts (9 with summer-type hypersensitivity pneumonitis of idiopathic pulmonar y fibrosis and 1 asymptomatic breeder), except for the 21-kd protein, which was detected in 10 patients with PBD and 1 asymptomatic breeder. The stimu lation indices of PBMCs determined by using proteins electroeluted from the 9 bands were higher in patients with PBD than in the 10 control patients. The 21-kd protein was separated into 5 spots by 2-dimensional electrophores is; these spots were all reactive with BAL fluid from patients with PBD as determined by immunoblotting. The sequence of the 21-kd protein had 57% ide ntity to a Saccharomyces cerevisiae chromosome X reading frame. A synthetic peptide, derived from the amino acid sequence of the N-terminal of the nat ive protein, induced significant proliferation of PBMCs obtained from 5 pat ients with PBD, but not with PBMCs obtained from control patients. Conclusion: The 21-kd protein is the only protein that identified individua ls exposed to pigeons by immunoblotting. Only PBMCs from patients with PBD showed significant proliferation to the 21-kd protein and to the synthetic peptide on the basis of the N-terminal sequence of the native peptide. The 21-kd protein will be an important antigen for studies on the epidemiology, diagnosis, and pathogenesis of PBD.