Model Construction of the Antibody Library of the Differential Proteome

• Main Authors: Sze-Han Chien, 錢思翰
• Other Authors: 莊榮輝
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/36523460522984801028

碩士 === 國立臺灣大學 === 生化科技學系 === 100 === Köhler and Milstein reported a novel technique by fusing B cell with myeloma cell and producing hybridoma cell lines which secreted monoclonal antibodies. Starting from this idea, we proposed a concept of massive antibody production by immunizing the total water-soluble proteins from an organism to yield antibody library against the whole proteome rather than immunizing single antigen to obtain single mAb. However, this approach consumed time, and needed huge human and material resources. Subsequently, Tsai proposed an alternative approach for differential antibody library, which focused on the different antigens of two comparable proteomes. After the immunization and cell fusion, hybridoma cells were screened using flow cytometer by the differences of the proteins samples. To validate this new approach, we focused on the maternal to zygotic transition (MZT) which is an essential period in early embryonic development. We took the earlier 128-cell stage and the sphere-cell stage of zebrafish embryo development as the two periods for comparison. In a preliminary test, an established hybridoma line was isolated successfully using this process. Then we immunized the embryonic total proteins in mice. After cell fusion, flow cytometer sorted out cells which produced antibody recognizing the fluorescent-labeled antigens shared by the two proteomes (negative screening). The remaining cells might produce antibodies which could bind to the differential proteins between the two proteomes. However, we can not harvest cell line producing useful antibody. Owing to this result, we then set up model test-2 using two sets of defined protein mixture as the antigens: (1) vitellogenin and low molecular weight markers, and (2) phytochelatin synthase and low molecular weight markers. After the cell fusion, positive selection was performed first to pick the high antibodies-secreting cell lines out, and then negative screening to remove the common cell lines subsequently. Results revealed that positive selection did identify antibodies-secreting cells, but we can not isolate differential cells in negative selection. Finally, we took zebrafish embryo proteins as the antigens to test its application in complex proteome, but still can not obtain the differential cell distribution. As a result, we should improve the whole preparation system.