Development of fluorescent-antibody reagents for demonstration of Pseudallescheria boydii in tissues

We prepared fluorescent-antibody reagents to detect and identify Pseudallescheria boydii in tissue. Antisera to broken mycelium and condidia (particulate antigens) and to culture filtrates (soluble antigens) of P. boydii were produced in rabbits. Antisera and globulin fractions of the antisera were labeled with fluorescein isothiocyanate and evaluated for their ability to stain P. boydii in tissues. The conjugates were first tested with cultures of 25 P. boydii isolates and 26 heterologous fungi that are morphologically similar to P. boydii in tissues. Labeled antibody to the soluble antigens reacted only weakly with cultures of P. boydii. Labeled antibody to particulate antigens brightly stained all P. boydii isolates and cross-reacted with Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Fusarium oxysporum, Fusarium solani, and a Scopulariopsis sp. The cross-reacting antibodies were removed by adsorption with A. fumigatus and F. oxysporum. The adsorbed conjugates stained P. boydii in Formalin-fixed tissues from experimentally infected mice and in Formalin-fixed tissues from humans and animals with natural infections. They did not stain the heterologous fungi in tissues.