Systemic candidiasis is the leading fungal bloodstream infection, and its incidence has been on the rise. Recently, Candida parapsilosis has emerged as an increasingly prevalent fungal pathogen, but little is known about its antigenic profile. Hence, the current work was performed to discover immunogenic proteins of C. parapsilosis using serological proteome analysis.
The Candida species are the most common fungal pathogens of systemic candidiasis. The diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, development of diagnostic assays to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry using specific monoclonals and polyclonals will be useful. Inbred Balb/c mice were immunized with C. albicans antigens, and blood was checked for the presence of reactive antibodies using ELISA. Fusion was performed using the harvested spleen cells and NS1 myeloma cells, and the clones were screened for the presence of antibody producing hybrid cells by dot-blot. Western blot analysis showed that the L2D10 monoclonal antibody was reactive against the antigens with molecular weight of 20 kDa. Experimental systemic candidiasis in mice was induced through intravenous injection of C. albicans and all the vital organs were collected for immunohistochemistry study. The monoclonal antibody reacted to surface epitopes on the yeast cells, germ tubes, and hyphae, and to immune complexes. It was used with the polyclonal antibody in a sandwich ELISA for the detection of circulating antigens in experimental candiadiasis in mice. Antibody levels were also determined using the ELISA method, and the antibody levels of C. albicans infected mice were increased compared with uninfected animals. The monoclonal antibody was used in immunoperoxidase and immunofluorescence techniques for the detection of fungal infection in tissue sections and was found to be more sensitive than conventional periodic acid Schiff or silver staining techniques. This monoclonal antibody may serve as potential primary capture antibodies for the development of a rapid diagnostic test for human systemic fungal infection.
Candida parapsilosis has emerged as one of the most common causes of bloodstream infection worldwide. The diagnosis of invasive candidiasis etiological agents to the species level remains a laboratory and clinical challenge. Thus, specific monoclonal antibodies to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry would be very useful. Inbred Balb/c mice were immunized with C. parapsilosis antigens, and blood was checked for the presence of reactive antibodies using ELISA. Fusion was performed using the harvested spleen cells and NS1 myeloma cells, and the clones were screened for the presence of antibody producing hybrid cells by dot-blot. The 1B11 clone secreted IgG2a monoclonal antibody that was reactive with the C. parapsilosis antigen at MW of 59 kDa and cross-reacted with C. tropicalis but not with other fungal and bacterial antigens tested. Another 3D1 clone secreted IgG1 monoclonal antibody that was reactive with C. parapsilosis antigen at MW of 30 kDa. The 3D1 monoclonal antibody was found to be species specific. Experimental systemic candidiasis in rats was induced through intravenous injection of C. parapsilosis, and all the vital organs were collected for immunohistochemistry study. These monoclonal antibodies were reactive against surface epitopes on the yeast cells, pseudohyphae, and immune complexes in tissue sections. Sandwich ELISAs using these antibodies were developed and were able to detect circulating antigens in experimental candidiasis in rats at 0.2 μg/μL. These monoclonal antibodies may have potential as primary capture antibodies for the development of rapid diagnostic test for human systemic fungal infection.
The serological responses to Cryptococcus neoformans proteins of blood donors and HIV patients with active cryptococcosis from a tropical region were investigated in this study. Exposure to C. neoformans, an organism ubiquitous in the environment, contributes to the antibody responses observed in the blood donors. IgG responses to cryptococcal proteins were stronger than IgM responses in most sera tested in this study. A 53-kDa cryptococcal protein fragment was identified as the most immunoreactive protein on the IgM immunoblots of both blood donors and patients. Overall, there was no obvious difference in IgG responses of patients when compared with blood donors. Some immunogenic protein fragments (27.5, 76, 78 and 91.5 kDa) were detected at least two times more frequently on IgM immunoblots of patients compared with those of blood donors. It is yet to be investigated whether the proteins identified in this study may have any potential to be used as biomarker for cryptococcosis.