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研究生(外文):Shu-Ching Chang
論文名稱(外文):Generation of anti-IL13 antibodies for diagnostic agent by LAE technique
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介白素13 (IL-13) 是由第二型輔助性T淋巴球分泌的一種細胞素,參與調節一些免疫反應,例如在氣喘、過敏、皮膚性過敏及發炎症狀裡血液中的IL-13濃度會增加。此外,IL-13可促進B細胞分化,使B細胞產生IgE,進而調節一些與發炎前期相關的細胞激素產生,並可增加MHCⅡ (major histocompatibility complex Ⅱ) 分子抗原呈現。這些臨床觀察意味著血液中IL-13含量可以作為一些免疫疾病診斷之指標。本研究期望製造抗IL-13之抗體,以作為開發快速檢測細胞激素之抗體晶片的材料。根據結構模型,選擇位於IL-13分子表面之不同區段作為抗原決定區 (epitopes),利用線性排列重複抗原技術,製造含線性重複抗原決定區之多胜肽 (linear array epitope),並與綠膿桿菌外毒素A接受器結合區(PEDIa)或麩胱甘肽轉移酵素(GST)結合成融合蛋白。以此純化之融合蛋白作為免疫原,免疫實驗兎與小鼠以刺激體液性免疫反應,並在ELISA檢測發現血清中確實存在抗IL-13不同抗原辨識區之抗體。同時為了製造具高度效價及穩定性之抗體,也利用融合瘤技術,選擇合適效價之小鼠脾臟以製造抗IL-13之單株抗體。目前,已成弗N四段抗原決定區,運用線性排列重複抗原技術得到的免疫原進行兔子免疫後,得到抗IL-13的多株抗體。在所得的抗血清中,發現以IL13-A10、IL13-D7、IL13-E9為抗原所產生之抗體,其分辨原態的IL-13能力皆可達0.5 ng以上。 而在單株抗體製備方面,已完成抗PE-A10與抗PE-E9之融合瘤單株化,並冷凍保存。未來,會進行抗體的純化,將抗體作原態IL-13辨認之分析,並且製備多量抗體以作為細胞激素抗體晶片的材料來源。
Interleukin-13 (IL-13) is a cytokine produced by T helper-2 lymphocytes and plays an important role in regulating several immune responses. The serum concentration of IL-13 increased in asthma, allergy, atopy and inflammatory conditions. It can promote B cell proliferation, induce B cells to produce IgE, down-regulate the production of proinflamatory cytokines and enhance the expression of class II MHC antigens. All these clinical observations implied that the serum concentration of IL-13 could be used as a diagnostic indicator for immunological disorders. We proposed to produce the anti-IL13 antibody, which could detect sub-concentration of IL-13 and be utilized for rapid diagnosis. Base on structural modeling, several different regions that exposed at the molecular surface were selected as antigenic targets. The linear array epitope (LAE) technique was employed to generate DNA fragments encoding for LAE antigen in fusion with Pseudomonas exotoxin domain Ia (PEIa) or glutathione S-transferase (GST). The purified fusion proteins were used to immunize rabbits. Antibodies against IL-13 epitopes were proved to be specific from ELISA assays. So far, we already have generated antigen of IL-13 epitopes by LAE technique, and taken the antigen to immunize rabbit to get the polyclonal antibodies. In these antisera, we found that antibodies against IL13-A10, IL13-D7, and IL13-E9 recognized the minimum quantity of native form IL-13 was 0.5 ng. Furthermore, in order to generate antibodies with high titer and stability, we prepared to generate monoclonal antibodies against IL-13 with hybridoma technique, and accomplished the monoclonal antibody against PE-A10 and PE-E9. The hybridoma cell lines were kept by freezing. In the future, we’ll purify antibodies of ascites to be the material of the antiboby chip.
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