Purification and Functional Analysis of Monoclonal Antibodies Protection Against C. auris Invasive Infection
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Disseminated candidiasis is the leading cause of life-threatening fungal infections in humans. This is especially the case in immunocompromised individuals, hospitalized patients that include intensive care unit patients. Despite the availability of antifungal therapy, crude mortality in the last decade has remained unacceptably high. The infection is caused by multiple species of the fungal genus Candida with C. albicans being the most common. Altogether, C. albicans, C. tropicalis and C. glabrata, cause >90% disseminated candidiasis in humans. Of particular concern, the “superbug” C. auris is a multi-drug resistant, health care-associated fungal pathogen, and has recently emerged as the first fungal pathogen to cause a global public health threat. Xin lab has identified and isolated a panel of monoclonal antibodies (mAbs) specific for Candida cell surface peptides, and the mAbs provide protection in mice against disseminated candidiasis caused by medically important Candida species, including C. auris. The goal of this study was to produce these mAbs by cell culturing hybridoma cells, adapt cells to CELLline flasks, and purify mAbs by using a Centricon device. Cell culturing is a useful technique in producing monoclonal antibodies in large quantities and in controlled conditions. The hybridoma cells were cultured in a nutrient rich media and were gradually adapted to serum free media. Once adapted, the cells were transferred to CELLline culture flasks where the cells were able to grow at high concentrations. Cells were harvested periodically to obtain each cell line’s supernatant containing mAb. The supernatants of hybridomas were then purified using centrifugal filtration and dialysis. Next, each mAb supernatant was isotyped to determine antibody subclass identity. Finally, the purified mAb endpoint titers and concentrations were determined with ELISA assays. ELISA, fluorescence staining and Flow cytometric analysis were performed to validate the binding of each mAb to specific Candida cell wall peptides. We conclude that the Immunoassay results show evidence of high functional titers of each peptide specific mAb, which provides protection against C. auris infections in mice. Antifungal antibodies could provide long-awaited novel therapies for use alone or in combination with antifungal agents.
Recommended Citation
Rodriguez, Claudia E., "Purification and Functional Analysis of Monoclonal Antibodies Protection Against C.
auris Invasive Infection" (2019). Summer Research Internship Program. 30.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/30
Purification and Functional Analysis of Monoclonal Antibodies Protection Against C. auris Invasive Infection
LSU Health Medical Education Building
Disseminated candidiasis is the leading cause of life-threatening fungal infections in humans. This is especially the case in immunocompromised individuals, hospitalized patients that include intensive care unit patients. Despite the availability of antifungal therapy, crude mortality in the last decade has remained unacceptably high. The infection is caused by multiple species of the fungal genus Candida with C. albicans being the most common. Altogether, C. albicans, C. tropicalis and C. glabrata, cause >90% disseminated candidiasis in humans. Of particular concern, the “superbug” C. auris is a multi-drug resistant, health care-associated fungal pathogen, and has recently emerged as the first fungal pathogen to cause a global public health threat. Xin lab has identified and isolated a panel of monoclonal antibodies (mAbs) specific for Candida cell surface peptides, and the mAbs provide protection in mice against disseminated candidiasis caused by medically important Candida species, including C. auris. The goal of this study was to produce these mAbs by cell culturing hybridoma cells, adapt cells to CELLline flasks, and purify mAbs by using a Centricon device. Cell culturing is a useful technique in producing monoclonal antibodies in large quantities and in controlled conditions. The hybridoma cells were cultured in a nutrient rich media and were gradually adapted to serum free media. Once adapted, the cells were transferred to CELLline culture flasks where the cells were able to grow at high concentrations. Cells were harvested periodically to obtain each cell line’s supernatant containing mAb. The supernatants of hybridomas were then purified using centrifugal filtration and dialysis. Next, each mAb supernatant was isotyped to determine antibody subclass identity. Finally, the purified mAb endpoint titers and concentrations were determined with ELISA assays. ELISA, fluorescence staining and Flow cytometric analysis were performed to validate the binding of each mAb to specific Candida cell wall peptides. We conclude that the Immunoassay results show evidence of high functional titers of each peptide specific mAb, which provides protection against C. auris infections in mice. Antifungal antibodies could provide long-awaited novel therapies for use alone or in combination with antifungal agents.
Comments
Mentor: Dr. Hong Xin MD, PhD, Department of Microbiology, Immunology, and Parasitology