Preparation and Application of Monoclonal Antibodies Against Human Prothymosin Alpha

• Main Authors: Chou, Chin-Hung, 周錦洪
• Other Authors: Wu Chao-Liang
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/13120918108539278698

碩士 === 國立成功大學 === 生物化學研究所 === 85 === Prothymosin a (ProTa) is an unusual acidic protein (PI=3.5) that widely distributes in mammalian tissue. It is a kind of thymic hormones associated with cell proliferation and has some immunomodulating properties. The relation of ProTa to proliferation has been well documented. ProTa mRNA expression is correlated to the proliferative activity of T-cells. In serum deprived NIH3T3 cells when they were stimulated to proliferate, ProTa mRNA levels were also induced. An increased concentration of ProTa in cancer tissue was reported. ProTa can restore the human mixed lymphocytic response (MLR) in patients of autoimmune diseases and enhance human and murine major histocompatibility complex (MHC) class II surface antigen expression. However, the mechanisms and real biological functions of ProTa are not very clear. In order to further study its biochemical properties, we produce monoclonal antibodies against human ProTa. The recombinant human ProTa proteins were obtained from the E. coli strain BL21(DE3)pLysS transformed with ProTa plasmid and purified by high salt/phenol extraction and recovered by enthanol precipitation. The SDS contamination in purification procedure was eliminated by re-extraction with n-butanol. After amino acid analysis and biological activity assay, we immunized female BALB/c mice with recombinant human ProTa proteins. The splenocytes were fused with NS-1 myeloma cells to prepare hybridomas. Six hybridoma cell lines which produced monoclonal antibodies against ProTa were obtained. Using one of the anti- human ProTa monoclonal antibodies, we developed a competitive ELISA system to quantify ProTa amount in urine samples of bladder cancer patients. The results indicate a significant difference of ProTa amount in urine between bladder cancer patients and normal healthy people.