三聚氰胺单克隆抗体研制及应用研究

发布时间：2018-05-29 18:28

本文选题：三聚氰胺 + 单克隆抗体　；参考：《重庆理工大学》2010年硕士论文

【摘要】： 三聚氰胺(Melamine,Mela)是一种化工原料,由于其含氮量高达66.6 %,常被非法添加到牛乳及饲料中,严重威胁人类和动物的生命安全。目前的检测方法大多需要专门的仪器设备并且操作繁琐,已无法满足现场快速检测的要求。因此,研究和建立一种方便、安全和快速的检测方法意义深远。本实验利用蛋白质连接技术合成了人工抗原,通过杂交瘤技术制备了抗Mela单克隆抗体,利用金标免疫层析技术成功研制了Mela金标免疫层析快速检测试剂盒,该试剂盒的灵敏度、特异性、稳定性等均符合检测要求,其规模化筛选特点更适用于常规检测需要,具有重要的现实意义和极高的商业价值。首先以多元酸酐与混合酸酐联用法将半抗原Mela分别与载体牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联制成人工抗原,以Mela-BSA免疫BALB/c小鼠,免疫小鼠脾细胞与SP2/0细胞进行融合,获得杂交瘤细胞。以Mela-OVA作为包被抗原用ELISA方法对杂交瘤细胞进行筛选,采用有限稀释法,经过对杂交瘤细胞的克隆和亚克隆,获得1株能稳定分泌抗Mela单克隆抗体的杂交瘤细胞株H2C6。以间接ELISA方法测定腹水单抗效价为1:204800,免疫层析实验(GICA)鉴定单抗与Mela特异性结合;H2C6单抗Mela的半数抑制浓度(IC50)为4.15μg/mL,与三聚氰酸、氯霉素、青霉素、链霉素、磺胺类药物等没有交叉反应;该细胞株体外多次传代培养和反复冻存复苏后抗体分泌稳定。采用柠檬酸三钠还原法制备直径20nm的胶体金并标记抗Mela单克隆抗体作为检测试剂,单抗加入量为20μg/mL,标记pH控制在9.0左右,以1% PEG20000作稳定剂。采用美国Millipore公司的FB-08玻璃纤维膜作为金标结合垫,Sartorius公司的CN-140硝酸纤维素膜做为层析膜制成金标免疫层析试剂盒,试剂盒的检测灵敏度为1μg/mL,在室温干燥条件下可保存18个月以上。本实验研制的胶体金免疫层析试剂盒最大的优点是无需借助检测仪器,检测快速、特异、灵敏、简便,5min内可出检测结果,适用于对Mela残留进行现场检测。本研究也为其他动物药物残留检测胶体金试纸的研制提供了借鉴。
[Abstract]:Melamine (melamine) is a kind of chemical raw material. Because of its nitrogen content as high as 66.6, it is often illegally added to milk and feed, which seriously threatens the safety of human and animal life. At present, most of the detection methods need special instruments and equipments, and the operation is tedious, which can not meet the requirements of field rapid detection. Therefore, it is of great significance to study and establish a convenient, safe and fast detection method. In this experiment, artificial antigens were synthesized by protein-binding technique, monoclonal antibodies against Mela were prepared by hybridoma technique, and the rapid detection kit for Mela gold-labeled immunochromatography was successfully developed by using gold-labeled immunochromatography, and the sensitivity of the kit was obtained. The specificity, stability and so on are in line with the requirements of detection, and the characteristics of large-scale screening are more suitable for routine detection, which has important practical significance and high commercial value. At first, the hapten Mela was conjugated with the carrier bovine serum albumin (BSA) and ovalbumin (ovalbumin) to form artificial antigens by using polyanhydride and mixed anhydride respectively. BALB/c mice were immunized with Mela-BSA, and spleen cells were fused with SP2/0 cells. Hybridoma cells were obtained. The hybridoma cell line H2C6 was obtained by using Mela-OVA as the coating antigen and ELISA method. The hybridoma cell line H2C6 was obtained by using the limited dilution method and the cloning and subcloning of the hybridoma cells. Indirect ELISA assay was used to determine the titer of ascites monoclonal antibody (1: 204800). The half inhibitory concentration (IC50) of McAb and Mela specific binding mAb Mela was 4.15 渭 g / mL with tripolycyanic acid, chloramphenicol, penicillin and streptomycin, and the specific inhibitory concentration of Mela was 4.15 渭 g 路mL ~ (-1) with tripolycyanic acid, chloramphenicol, penicillin and streptomycin. There was no cross reaction between sulfanilamides and other drugs, and the antibody secretion of the cell line was stable after repeated cryopreservation and resuscitation. Colloidal gold with diameter 20nm was prepared by trisodium citrate reduction method and labeled with monoclonal antibody against Mela as detection reagent. The amount of monoclonal antibody was 20 渭 g / mL, the labeling pH was about 9.0, and 1% PEG20000 was used as stabilizer. The gold standard immunochromatographic kit was made by using the FB-08 glass fiber membrane of Millipore Company as the gold standard binding pad and the CN-140 nitrocellulose membrane of Sartorius Company as the chromatography membrane. The sensitivity of the kit was 1 渭 g / mL, and the kit could be preserved for more than 18 months under the condition of room temperature drying. The greatest advantage of the colloidal gold immunochromatographic kit developed in this experiment is that it is rapid, specific, sensitive and simple to detect Mela residues in 5 minutes without the aid of detection instruments. It is suitable for the field detection of Mela residues. This study also provides a reference for the development of colloidal gold test paper for the detection of drug residues in other animals.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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