Three Paracoccidioides brasiliensis antigens, namely a culture filtrate preparation, a somatic antigen and a mixture of equal parts of the two, were tested by two serological techniques against sera from patients with paracoccidioidomycosis, and in an in vivo delayed hypersensitivity model in mice. The antigen mixture was more sensitive than the two individual antigens for the evaluation of the humoral and cellular immune response to P. brasiliensis, both in man and in experimental animals.Immunodiffusion is the most widely used test for the diagnosis and monitoring of paracoccidioidomycosis. In this test, most investigators have used either a somatic antigen or a culture filtrate preparation of Paracoccidioides brasiliensis, with varying results regarding sensitivity and specificity [ 10].At the IV International Meeting on Paracoccidioidomycosis held in Caracas, in 1989, it was demonstrated that the combined use of different P. brasiliensis antigens could increase the sensitivity of immunodiffusion for the diagnosis of the disease, as well as for monitoring the effect of treatment and post-therapy follow-up [ 1]. However these serological tests have to be performed separately for individual antigens. In the present study we have evaluated the sensitivity of three P. brasiliensis antigens, namely a culture filtrate preparation, a somatic antigen and a mixture of equal parts of the two, in two serological methods and in a delayed hypersensitivity test, for the detection of humoral and cellular immune responses to P. brasiliensis.The three following antigens, prepared from the same pool of P. brasiliensis strains (strains 18, 113 and 192), were used.Culture filtrate. Viable yeast forms were inoculated into flasks containing a synthetic culture medium [8]. After incubation at 37°C with shaking for 7 days, the fungal cells were killed with thimerosol (Sigma). The cultures were centrifuged at 2000 rev. min-' for 10 rain and the pellets stored for the preparation of the somatic antigen. After filtration, the culture filtrates were pooled into a single antigen batch, concentrated, dialysed and lyophilized.Somatic antigen. The culture pellets were mixed, resuspended in sterile saline and sonicated using phenylmethylsulfonyl fluoride (Sigma) as a protease inhibitor [7]. The sonicated
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