甲基安非他命生物共軛物之合成及其多株抗體之生產

Studies on the Synthesis of Methamphetamine-Protein Bioconjugates and Production of Polyclonal Antibodies against Methamphetamine

本研究目的在於自行合成甲基安非他命(methamphetamine; Meth)與蛋白質之生物共軛物(bioconjugates)來腹腔免疫ICR小鼠，以生產Meth之多株抗體(polyclonal antibodies against methamphetamine; anti-Meth PAbs)。由於Meth之分子量很小（約149.233g/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Meth形成生物共軛物，藉由大分子蛋白誘導小鼠體內產生Meth之抗體。多株抗體製作過程為將高度免疫hyperimmune）之ICR小鼠，注射0.5mL 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HitrapTM rProtein A column來進行多株抗體之初步純化，並進一步測定anti-Meth PAbs之效價(titer)以及進行蛋白質定量工作。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Meth與BSA之生物共軛物(Meth-BSA)當作免疫原(immunogen)進行小鼠免疫工作；另外，Meth與Ser之生物共軛物(Meth-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5.68 mg/mL多株抗體（含anti-Meth and anti-BSA PAbs），並加以冷凍乾燥保存。

The aim of this study was to synthesize methamphetamine (Meth)-protein bioconjugates and produce polyclonal antibodies (PAbs) against Meth (anti-Meth PAbs). ICR mice were intraperitoneally immunized with Meth-protein bioconjugates every two weeks. Because the molecular weight of Meth is very small, it is difficult to induce production of mice antibodies. The Meth molecules were conjugated with bovine serum albumin (BSA) and sericin (Ser), respectively. Hyperimmune ICR mice produced anti-Meth PAbs after injected with 0,5 mL pristane, and injected with NS-l myeloma cells two weeks later. Ascites and serum were collected from ICR mice, but before the mice had difficulty moving, and anti-Meth PAbs was purified by using Hitrap(superscript TM) rProtein A column. In this study, the drug-proteins bioconjugates had been successfully produced. Meth-BSA bioconjugates were as the immunogen for mice immunization and Meth-Ser bioconjugates were as the antigen for Enzyme-Link Immunosorbent Assay (ELISA). Finally, the production and primary purification of anti-Meth PAbs were performed. The concentration of PAbs obtained was 5.68 mg/mL. Then, the anti-Meth PAbs were stored by freeze-dry method.