Production of a monoclonal antibody against a yeast secreted antigen of Penicillium marneffei.

ABSTRACT

Monoclonal antibody against P. mameffei yeast secreted antigen was produced in order to develop a serological test for penicilliosis marneffei. The yeast form of P. marneffei was cultured in brain heart infusion broth at 37 degrees C for 7 days. A secreted antigen was prepared, partially purified from culture supernatant and subsequently immunized in a BALB/c mouse. Mouse monoclonal antibody was produced from immune spleen cells by a standard hybridoma technique. Specificity of the obtained monoclonal antibody was assessed with yeast secreted antigens for P. mameffei, C. alblicans, C. neoformans, and H. capsulatum by an indirect ELISA. Three of 46 hybrid clones (1 F1, 2G5, and 3G4) reacted positively with P mameffei secreted antigen. 1 F1 and 3G4 were cloned by two rounds of limiting dilution. Partially purified monoclonal antibody and rabbit polyclonal antibody against P. marneffei yeast secreted antigen were used to develop a double antibody sandwich ELISA to detect P. marneffei antigen in plasma or serum samples of 7 patients with penicilliosis marneffei and 5 healthy controls. The sandwich ELISA developed using monoclonal antibody as a capture antibody and rabbit polyclonal antibody as a detector was able to detect P. marneffei antigen in all the plasma and serum samples of penicilliosis marneffei patients, while negative in all the healthy controls. Thus, the monoclonal antibody produced in the present study appeared to be highly specific for P. marneffei and the double antibody sandwich ELISA developed using monoclonal and polyclonal antibodies against the yeast secreted antigen of P. marneffei showed a strong potential for the diagnosis of penicilliosis marneffei.