- 學位論文
卵巢癌相關OVTA-1-1002抗原小鼠單株抗體的製備及定性
Production and characterization of mouse monoclonal antibody specific reactive with ovarian cancer associated OVTA-1-1002 antigen
摘要
為提高卵巢癌的早期診斷準確率，篩選具高度特異性、敏感性的卵巢癌的生物標記及發展檢驗方法是迫切且急需的。本實驗室已利用卵巢癌病人腹水的抗體CA502，從phage diplay peptide library篩選出OVTA-1基因所表現的蛋白質，是卵巢癌腫瘤細胞相關的抗原蛋白。本研究進行OVTA-1-1002 卵巢癌相關的小鼠單株抗體製備研究。首先，利用大腸桿菌表現OVTA-1-1002蛋白質並以SDS-PAGE確認OVTA-1-1002抗原蛋白質分子量，並利用MicroBCA測量OVTA-1-1002蛋白質濃度，之後免疫注射Balb/c小鼠並探討小鼠血清中Anti-OVTA-1-1002多株抗體隨免疫反應時間表現量變化，再取出小鼠脾臟細胞與骨髓癌細胞融合產生Anti-OVTA-1-1002單株抗體，並以MicroBCA測定純化後Anti-OVTA-1-1002單株抗體的濃度以及探討抗原抗體最佳反應濃度。Anti-OVTA-1-1002單株抗體抗原結合利用競爭型酵素免疫分析法評估，並確認Anti-OVTA-1-1002單株抗體分型IgG1 kappa light chain。
並列摘要
To improve the early detection of ovarian cancer, the screening highly specificity and sensitivity ovarian cancer biomarker is urgent and the development of ovarian cancer diagnosis is highly demanded. Our laboratory used phage display random 7-mer peptide library system to screen ovarian cancer associated antigens. It probed with ascitic CA502 antibody by enzyme-linked immunosorbent assay and the novel OVTA-1 gene expressed proteins were identified as ovarian cancer associated antigens. In this study, we produced and characterized the mouse monoclonal antibody which was specific reactive with ovarian cancer associated OVTA-1-1002 antigen. First, E.coli was used for the expression of OVTA-1-1002 antigen. The molecular weight and concentration of OVTA-1-1002 antigen was determined by SDS-PAGE and MicroBCA, respectively. Then, the Balb/c mouse was immunized with the OVTA-1-1002 antigen. The effect of immune response time was investigated. The splenocytes of mouse immunized with the OVTA-1-1002 antigen were collected and fused with myeloma cell for the production of Anti-OVTA-1-1002 monoclonal antibody. The concentration and binding curve were evaluated. The isotype of anti- OVTA-1-1002 monoclonal antibody is IgG1 kappa light chain.