Standardization and production of exoantigens of Paracoccidioides brasiliensis in NGTA medium
Assis, C. M.& Rodrigues, M. C.
Instituto Adolfo Lutz, São Paulo, SP. cmassis@ial.sp.gov.br
Paracoccidioides brasiliensis (Pb) is a dimorphic fungus that causes paracoccidioidomycosis, a common systemic mycosis in Latin America. The diagnosis is realized with the demonstration of the fungus by optical microscopy or by isolation in culture. It is accomplished by serological tests. The purpose of this study is the production of crude antigens from Pb to be used in serological tests. Cells of Pb 113 were obtained from cultures in Fava Netto agar medium, at 36ºC during five days. After growth of the cultures suitable viability and concentration were determined. Five ml of the suspension were inoculated into 200 ml of NGTA (neopeptone, glucose, thiamine and asparagine) medium and incubated at 36ºC, during 20 days, with agitation of 45 rpm. The cells were then killed with thimerosal 1:5000 and separeted from crude antigen by filtration and it was concentrated 10 times and kept at 4ºC. The obtained antigen was studied by double immunodiffusion (DI), crossing over immunoelectrophoresis (CIE), immunoelectrophoresis (IEF), complement fixation (CF), electrophoresis with SDS-PAGE, immunoblot (IB) methods with sera of pacients of several mycoses as paracoccidioidomycosis, histoplasmosis, aspergillosis, lobomycosis, other infections diseases as tuberculosis, leishmaniasis, healthy donors and hiperimmune sera of Paracoccidioides brasiliensis, Histoplasma capsulatum, Aspergillus fumigatus, Candida albicans. Results showed three lines of precipitation in double immunodiffusion test, five lines in immunoelectrophoresis test. Titles of 1:4 in DI test, 1:4 in CIE test, 1:256 in CF method and sera of patients with other diseases, heterologous antisera and sera of healthy donors presented no reaction. SDS-PAGE and IB showed different bands of glycoproteins between 19 to 105 kDa and better expression of gp43. Antigen of Pb in NGTA medium with dextrose and peptone for serological diagnostis of paracoccidioidomycosis was produced and standardized. As we showed this medium produced crude antingens with less contaminantion (protein and dextrose) and better visibility of bands of the glycoproteins. As compared with Negroni medium modified our results indicated the medium used in this study is cheaper and easier to work.
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