Monoclonal Antibody Production Against Candida Albicans And C. Parapsilosis And Immunopathological Changes In Experimental Infections In Rodents

Abstract

Candida spp. Are the most common fungal pathogens of systemic candidiasis. As there are pathognomonic signs or symptoms of systemic candidiasis, the diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, diagnosis assays to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry using specific monoclonals and polyclonals will be useful. Balb/c mice were immunized with C. albicans and C. parapsilosis and blood were checked for the presence of reactive antibodies using ELISA. Fusions were performed using the harvested spleen cells and NSI mouse myeloma cells and the clones were screened for the presence of antibodies producing hybrid cells by dot-blot. A total of forty and fifty clones producing monoclonal antibodies against C. albicans and C. parapsilosis respectively were obtained. Western blot analyses showed that the monoclonal antibodies against non heat-shocked (IgM) and heat-shocked (IgG3) C. albicans were reactive against the antigens with MW at 30 and 59 kDa. Experimental systemic candidiasis in mice and rats were induced through intravenous injection of C.albicans and C. parapsilosis. The liver, spleen, kidneys, brain, heart and lungs were collected for histopathological examination. Cystic lesions with large clumps of fungus consisting mainly of hyphae and some yeast were observed in the kidneys. Clusters of yeast cells were detected in multiple organs. The female mice and rats were more resistant to the C. albicans infection than the male counterparts. The rats were found to be more susceptible to C. albicans infection than Balb/c mice. C. parapsilosis was found to be less virulent than C. albicans. The animals infected with C. parapsilosis had mild infection in spleen and lungs only. However, all the majority of the organs in animals that succumbed to the C. albicans or C. parapsilosis infection had fungal invasion. Histopathhological examination revealed that all dead but no those surviving or control animals had mild to serve fungal colonization in brains. Fungal colonization in the brain may be a determinant factor for mortality in experimental systemic. Candidiasis. Two Isolates of fungal were obtained from blood culture on Sabouraud dextrose agar and were found to be of the same species with those used for the infection, based on restrictive length polymorphism and DNA alignment analysis. The monoclonal antibodies were purified, characterized and used in the immunoperoxidase and immunofluorescence techniques. The monoclonal antibodies reacted to surface epitopes on the yeast cells, germ tubes, hyphae and to immune complexes. The monoclonal antibodies obtained were used for the detection of antigens, together with the polyclonal antibody, in the experimental model and were found able to detect antigens present in the sera at 0.2µg/µL. Antibody levels were also determined using the ELISA method. The antibody levels against C. albicans or C. parapsilosis in infected mice or rats were increased compared with uninfected animals. The Circulating antigen level in the infected animals increased initially but declined subsequently. The cytokine levels (IL-4, IL-6, IL-10, IFNƴ and TNF) induced in the models were measured using flow cytometry. C. albicans and C.parapsilosis elicited different cytokine expression in mice and rats. The groups infected with C. albicans had higher IL-6 and TNF, but unchanged IL-4, IL-10 and IFNƴ levels when compared with uninfected mice. Elevated levels of TNF were seen in both mice and rats infected with C. albicans but not in those infected with C. parapsilosis. The level of IL-10 was lower for all the C. albicans and C. parapsilosis infected rodents compared with unifected rodents except for rats infected with C. albicans at the later stage of infection. In conclusion, monoclonal antibodies were produced and user for the detection of antibody, circulating antigen and immune complexes in the experimental systemic candidiasis model. These monoclonal antibodies may serve as potential primary capture antibodies for the development of rapid diagnostic test for human systemic Candida infection Fungal colonization in different organs and differential host immune responses against C. albicans and C. parapsilosis were shown in the present study.