Generation and Characterization of Polyclonal and Monoclonal Antibodies against Candida albicans Alpha-Enolase

• Main Authors: Po-Yen Liao, 廖柏彥
• Other Authors: 呂思潔
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/mgge42

碩士 === 臺北醫學大學 === 醫學科學研究所 === 102 === C. albicans is an opportunistic human pathogen, which colonizes at several sites including skin, oral, gastrointestinal track and vagina. C. albicans is also a major pathogen caused clinical candidemia of 50.4%. Candidemia is a fungal infection that can occur when Candida yeasts enter the bloodstream, is extremely rare in health people. In recent decades, due to a defective immune patient population such as organ transplantation, blood cancer, receiving immunosuppressive, or chemotherapy and AIDS patients increasing, Candidemia becomes important issue. Candidemia has a very significant morbidity and mortality. In the United States, Candida species are the fourth leading cause of nosocomial bloodstream infection, and the attributable mortality to be 49%. Alpha–enolase（ENO1） is a key glycolytic enzyme . C. albicans Alpha–enolase (CaENO1) can be detected in the sera of candidemia patient, and mutations on Candida alpha-enolase in C. albicans would inhibit cell growth. CaENO1 were used as an important marker protein in medical application. In this study, CaENO1 recombinant DNA transformed into E. coli, CaENO1 protein could be expressed and purified. After the chicken immunized with CaENO1 recombinant protein, it succeed to generate the polyclonal chicken IgY. These polyclonal antibodies could specifically bind to CaENO1 protein in Wesrern blot and ELISA. Following, we used phage display technology to construct two scFv（single chain variable fragment）antibody library (short linker and long linker, 2.4×106 and 1.36×107 , respectively ). After the fourth round of bio-panning, we selected a monoclonal antibody CaS1 with specific binding ability to CaENO1. In Western blot analysis, CaS1 antibody can specifitily recognize to alpha-enolase on C. albicans and Candida tropicalis. CaS1 antibody can bind to C. albicans surface alpha-enolase in flow cytometry analysis. CaS1 can also inhibit Candida albican growing on YPD agar plate and reduce Candida albican binding ability to oral epidermoid carcinoma cell line (OECM-1) cell. In vivo test showing that CaS1 can prolong survival of candidemia mice. CaS1 can inhibit C. albicans surface alpha-enolase binding to plasminogen by fibrin matrix-gel degradation analysis. According to the results, CaS1 antibodies could be used to detect and treat C. albicans infection in the future.