Candida Albicans Enolase

Invasive fungal infections have been ever increasing in recent years, and have merged as major causes of morbidity and mortality in immuncompromised patients. Candida albicans is the most common pathogen of all fungus. Enolase, an enzyme that catalyses the reversible dehydration of 2-phosphaglycerate to phosphoenolpyruvate, is an immunodominant antigenic component in disseminated Candida albicans infection. It is useful for clinical diagnosis and prevention of candidiasis, and illumination of pathogenic mechanism of Candida infection to study the functions and biological activity of the enzyme which is one of highly conserved proteins in cell plasma of Candida albicans.Firstly, the enolase of Candida albicans was isolated and identified. After a standard strain of Candida albicans was fermented on liquid medium for 24h at 37℃, yeast cell were collected. Lyticase and supersonic method were applied to disrupt the cell wall and membrane of Candida albicans. Saturated NH₄SO₄ solution was added to the supernant, and the precipitated material between 50 and 70% saturation was collected. The enolase was purified by DEAE-Sephadex A-50 gel column and ConA-Sepharose-4B gel column chromatography. The molecular weight of the protein determined by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 47KD. The enzymatic activity measured through glycolytic reaction chain in vitro was 2950.0u/ml at 30℃. The rate of protein obtained through this method of isolation is 6.o%.The purified enolase was used to immunize rabbit for stimulation of anti-enolase serum. The highest titer of polyclonal antibody was up to 1: 6400 with enzyme-linked immunoadsordent assay ( EL1SA) . The specificity of enolase antigen was demonstrated by western blot analysis with the serum from Candida albicans infected rabbit. In the same time, anti-whole cell protein and nonglycoprotein of Candida albicans sera were developed. There three kinds of antibody and the serum from Candida albicans infected rabbit were applied to detect the antigen of C. krusei, C guilliermondii, C. glabrata, C. tropic, S. cerevisiae, A. flavus and A. fumigatus in vitro. The anti-enolase antibody was superior to the two others in specificity.In C. albicans-infected immuno-normal mice and mice which had been immunosuppressed with cyclophosphamide peritoneally, We evaluated the immunoelectrophoresis technique using anti-enolase serum as antibody in diagnosis of candidiasis in comparison with blood culture. In the 3^rd day after infection, the antigen in some normal mice were detected with...