【作者】 赵海锋；

【导师】 甘一如；

【作者基本信息】 天津大学 ， 生物化工， 2007， 硕士

【摘要】 本文应用胶体金免疫层析技术进行吗啡(MOR)药物滥用的尿液及唾液检测研究。为了研制吗啡胶体金免疫层析试纸条,本研究利用吗啡偶联物抗原来制备单克隆抗体,采用微波加热方法制备20 nm胶体金,鉴定质量后标记单克隆抗体。将金标抗体喷涂在聚酯膜上,MOR-BTG包被在硝酸纤维素膜上,制成快速检测试纸条。首先本文在传统的微波加热法的基础上进行改进,制备胶体金。研究了不同的加热时间和还原剂对胶体金制备的影响,并将改进的微波法与传统微波法、电热套法和磁力搅拌法进行比较,结果表明,改进的微波法更适于制备单分散性小粒径的胶体金。本文还利用混合酸酐法将MOR和牛血清白蛋白(BSA)、牛甲状腺球蛋白(BTG)偶联,偶联物分别作为免疫抗原和包被抗原。再用免疫抗原MOR-BSA免疫得到抗吗啡单克隆抗体。最后通过比较不同的金标蛋白标记条件、不同的材料、聚酯膜上金标的喷涂量、抗原和二抗的包被浓度等对检测结果的影响,确立了最佳条件,成功建立了尿液和唾液联检吗啡的胶体金免疫层析方法。该法能在5分钟内完成对吗啡的检测,尿样和唾液的最小检测限分别为300 ng/ml、20 ng/ml,无需借助检测仪器;取样方便,检测时间段宽,适用于对吗啡进行现场快速检测。本研究也可为其它药物滥用检测试纸条的研究提供借鉴。更多还原

【Abstract】 A gold immunochromatographic assay(GICA) was applied to test for opiods of abuse in saliva and urine. In order to establish a one-step strip test for the detection of morphine, monoclonal anti-morphine antibody was prepared after antigen coupled. 20nm gold solution was prepared by microwave heating, and conjugated with the monoclonal antibody after identification. The conjugation solidified to polyester membrane, MOR-BTG was immobilised on nitrocellose membrane to prepare for kit.The method of microwave heating for preparation of gold solution was improved. Effections of heating time and reducer addition during preparation were explored. The result showed that the improved method of microwave was more siutable for preparation of small diameter gold particle.Morphine was coupled with BTG or BSA by mixed acid anhydride, to produce the immuno antigen and the coating antigen. Then the McAbs were attained with the conjugant was carried.Finally, the various factors and conditions of the immunochromatographic lateral-flow assay were explored, and the optimal reaction conditions of the assay were ascertained. GICAs for rapid detection of morphine in saliva and urine were successfully established. The GICA devices had the cut-off value of 300 ng/ml and 20 ng/ml respectively for morphine dissolved in saliva and in urine. The result could be obtained in 5 minutes. It was very suitable for rapid test for the morphine in saliva and urine. The method producing GICA for morphine could also be used as reference in the development of the test for abuse of other drugs.更多还原