| 摘要: | 愷他命 ( Ketamine ) 及氟硝西泮 ( Flunitrazepam, FM2 ) 原本作為醫療用途，具有麻醉和鎮靜安眠的功效，但在台灣成為主要的非法濫用藥物。其主要功能為麻醉、鎮定精神、速效的安眠作用，但因愷他命使用時會產生欣快感以及氟硝西泮的快速安眠效果，所以被用做非法用途。本研究的目的主要是愷他命及氟硝西泮單株抗體之生產，並將自行生產之抗體應用於奈米金免疫層析試片 (Nanoparticle-Gold Immunochromatographic strip) 之開發上。本研究所開發之試片可應用於警政單位進行初步篩檢或保護自身安全之樣品檢測，極具應用價值。
首先進行愷他命及氟硝西泮與蛋白質生物共軛物之結合，本實驗已合成出愷他命及氟硝西泮與胎牛血清蛋白之共軛物 (Ketamine-BSA、FM2-BSA) 以及愷他命及氟硝西泮與純蠶絲蛋白之共軛物 (Ketamine-Ser、FM2-Ser)，利用 Ketamine-BSA、FM2-BSA當作免疫原，Ketamine-Ser、FM2-Ser 當作抗原進行檢測。
接著進行愷他命及氟硝西泮單株抗體之生產與純化。實驗成功篩選出6株高效價之單株抗體融合瘤細胞株，分別為KF7、KG4、KD6、KC3、FB1及FE6，經鑑定其單株抗體同種型 ( isotype ) 全屬於IgG2a 重鏈及 κ 輕鏈。其中 KF7 單株抗體經大量生產後，以 Hitrap™ rProtein A Fast Flow Column 純化，經間接型 ELISA 分析抗體效價為1：15,625，且與絲膠蛋白及牛血清蛋白無交叉反應。
第二部分進行奈米金粒子 ( nanoparticle-gold ) 之合成工作。開發出的免疫層析試片能夠檢測愷他命。
本研究所製備之免疫層析試片，具有以下優點：1. 高度專一性；
2. 靈敏度高；3. 不需前處理；4. 所需樣品量少；5. 操作方便；
6. 檢測快速；7. 低成本；8. 沒有交叉反應；9. 可長期保存；10. 不
需要任何儀器之使用等優點。因此本實驗所製備之免疫層析試片極具應用價值。
Ketamine and flunitrazepam ( FM2 ) had a medical use, has a hypnotic effect of anesthesia and sedation, but in Taiwan as a major illicit drug abuse. Its main function is anesthesia, calm spirit, quick of hypnotic effect, but ketamine use produces euphoria and FM2 effects of the fast sleep, it was used for illegal purposes. The objective of this study was to produce monoclonal antibodies against Ketamine and FM2. These antibodies will be used for application in the development of a nanoparticle-gold immunochromatographic strip. This study will be used to the preliminary of selective sample in the police authorities of municipality.
Firstly, in order to synthesize Ketamine and FM2-BSA conjugation, this study has synthesized Ketamine and FM2-BSA and Ketamine and FM2-Ser bioconjugate. Moreover, production and purification of monoclonal antibodies against Ketamine and FM2 .
Then, this study was to produce and purification anti-Ketamine and FM2 mAbs. Six high-titer anti-Ketamine and FM2 mAb-producing hybridoma cell lines were obtained and designated KF7, KG4, KD6, KC3, FB1 and FE6. These isotypes of mAbs were identified as IgG2a heavy chain and κ light chain. For the purification of anti- Ketamine and FM2 mAbs, Hitrap™ rProtein A Fast Flow column was also used. The highest titer of anti-Ketamine mAb ( KF7 ) determined by indirect ELISA was 1:15,625, and no cross-reaction with sericin and BSA.
Secondly, the nanoparticle gold was made from the chloroauric acid (HAuCl4). This immunochromatographic strip could determine Ketamine. The advantages of self-assembled immunochromatographic strip was high specificity, high sensitivity, no pretreatment, low sample requirement, easy operation, rapid determination, low price, no cross-reaction, long-term preservation, no machine needed etc. |
