Immunochemical and subcellular localization of the 43 kDa glycoprotein antigen of Paracoccidioides brasiliensis with monoclonal antibodies.

Two monoclonal antibodies ST-7 (IgG1) and ST-8 (IgG2b), directed against a 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis were produced. It was possible to detect the gp43 by ELISA in amounts as low as 100 ng per well, and by Western blot about 300 ng were detected. Mild treatment of the gp43 with sodium metaperiodate did not alter its reactivity with ST-7 and ST-8, which suggests that these MAbs recognize peptide epitopes. Confirming the periodate oxidation data, the 38 kDa protein resulting from deglycosylation of the gp43 with trifluoromethanesulphonic acid (TMFS), was reactive with ST-7 and ST-8. Immunoelectron microscopy showed that the gp43 is stored inside large dense core vesicles, which flowed into the plasma membrane and extruded from cell membrane into the cell wall. Finally the antigen was secreted into the extracellular space as dense membrane-free material. Secretion of the gp43 occurred at scattered sites interspersed along the cell surface.