IMMUNOLOGICAL DATA OBTAINED OF Paracoccidioides brasiliensis EXOANTIGENS FROM CULTURES IN NGTA (NEOPEPTONE, GLUCOSE, THIAMINE AND ASPARAGINE) MEDIUM. PRODUCTION AND STANDARDIZATION OF ANTIGENS AND HIPERIMMUNESERA

ASSIS C. M.(1), RODRIGUES M. C.(2)

(1)Instituto Adolfo Lutz - São Paulo, SP, Brazil, (2)Department of Pathology,  FMUSP

Paracoccidioides brasiliensis (Pb) is a dimorphic fungus that causes paracoccidioidomycosis, a common systemic mycosis in Latin America. The diagnosis is performed with the demonstration of the fungus from clinical materials 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 serology. Yeast cells of Pb 113 were subcultured in NGTA agar medium by four times, 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  medium and incubated at 36ºC, during 20 days with agitation. The cells were then killed with thimerosal 1:5000 and separated from crude antigen by filtration, concentrated 10 times and kept at 4ºC. The obtained antigen was studied by gel immunodiffusion tests, complement fixation (CF), electrophoresis with SDS-PAGE and immunoblot (IB) methods with sera of patients of several mycoses as paracoccidioidomycosis, histoplasmosis, aspergillosis, lobomycosis, other infectious diseases, healthy donors and hiperimmune sera of Pb, Hc, Af and Ca. Results showed three lines of precipitation in double immunodiffusion and five lines in  immunoelectrophoresis tests. Titles of 1:4 in immunodiffusion, 1:4 in counterimmunoelectrophoresis and 1:256 in CF were obtained. 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. It was demonstrated that this medium produced crude antingens with less protein contaminantion and better visibility of bands of the glycoproteins. As compared with Negroni modified medium these studies indicate that NGTA medium used in this research is cheaper and easier to work, presenting good results in serology and production of antigens and hiperimmunesera.

KEY WORDS: paracoccidioidomycosis, antigens, hiperimmunesera, serology

CORRESPONDENCE TO:

Cezar Mendes de Assis, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355, 2º andar, sala 65, CEP 01246-902, Cerqueira César, São Paulo, SP. Email: cmassis@ial.sp.gov.br