Flunitrazepam生物共軛物之合成及其多株抗體之生產

Studies on the Synthesis of Flunitrazepam-Protein Bioconjugate and Production of Polyclonal Antibodies against Flunitrazepam

本研究之目的在於自行合成flunitrazepam (FM2)與蛋白質之生物共軛物(bioconjugates)並以其為免疫原來腹腔免疫ICR小鼠以生產FM2之多株抗體(polyclonal antibodies against FM2; anti-FM2 PAbs）。內於FM2分子量小（約313.3g/mol），不易在小鼠體內引起免疫反應，故本研究選用牛血清白蛋白(bovine serum albumin; BSA)以及絲膠蛋白(Sericin; Ser)，分別與FM2合成生物共軛物，藉由大分子蛋白誘導小鼠體內產生FM2之抗體。多株抗體製作過程為將高度免疫(hyperimmune)之ICR小鼠，汪射0.5 mL pristane後，再間隔兩週注射10^6個NS-1骨髓癌細胞。待小鼠腹腔漲大至行動困難時，收集ICR小鼠所產生之腹水及血清，利用Hitrap（上標 TM）rProtein A column來純化此多株抗體，並進步測定經初步純化之anti-FM2 PAbs之效價(titer)。本實驗工作首先成功合成出藥物與蛋白質之生物共軛物，其中將FM2與BSA之生物共軛物(FM2-BSA)常作免疫原(immunogen)進行小鼠免疫工作；另外，FM2與Ser之生物共軛物(FM2-Ser)當作酵素連結免疫吸附分析法(Enzyme-linked immunosorbent assay; ELISA)檢測系統中所用之抗原(antigen)，以避免測到腹水中之anti-BSA抗體效價，而造成誤差。最後生產並初步純化出9.49mg/mL多株抗體（含anti-FM2及anti-BSA），而後加以冷凍乾燥保存。

The aim of this study was to synthesize flunitrazepam (FM2) -protein bioconjugates and produce polyclonal antibodies (PAbs) against FM2 (anti-FM2 PAbs). ICR mice were intraperitoneally immunized with FM2-protein bioconjugates every two weeks. Because the molecular weight of FM2 is very small, it is difficult to induce antibodies production for mice. The FM2 molecules were conjugated with bovine serum albumin (BSA) and sericin (Ser), respectively. Hyperimmune ICR mice produced anti-FM2 PAbs after injected with 0.5 mL pristane, and injected with NS-1 myeloma cells two weeks later. Ascites and serum were collected from ICR mice, but before the mice had difficulty moving, and anti-FM2 PAbs was purified by using Hitrap(superscript TM) rProtein A column. In this study, the drug-proteins bioconjugates had been successfully produced. FM2-BSA bioconjugate was as the immunogen for mice immunization and FM2-Ser bioconjugate was as the antigen for Enzyme-link Immunosorbent Assay (ELTSA). Finally, the production and primary purification of anti-FM2 PAbs were performed. The concentration of PAbs obtained was 9.49 mg/mL. Then, the anti-FM2 PAbs were stored by freeze-dry method.