金刚烷胺单克隆抗体的制备及ELISA检测方法的建立

发布时间：2018-08-28 17:10

【摘要】：金刚烷胺(Amantadine,AMT)属于三环胺类,是最早用于治疗流感的抗病毒药。在畜禽养殖中,主要用于治疗和预防鸡禽流感,以及猪传染性胃肠炎的防治,但是其致精神异常的副作用以及日益增强的耐药性对畜产品消费者可能造成的危害越来越引起关注。因此,我国农业部560号公告中已明令禁止金刚烷胺和金刚乙胺等抗病毒药物用于畜禽养殖业,美国FAD也已明确禁止在畜禽养殖中使用此类药物。检测金刚烷胺的残留可采用传统的仪器检测方法,主要有液相色谱法、气相色谱法、液相色谱-串联质谱法、超高液相色谱-串联质谱法、亲水作用色谱-串联质谱法以及电位滴定法等。然而,传统的仪器检测方法不仅需要昂贵的仪器设备,复杂的样品处理过程还需要专业的技术人员进行操作,这些不利因素使这些检测方法不适用于现场高通量的样品检测。ELISA检测方法基于抗原抗体特异性结合,具有高度的选择性和灵敏度,检测方法成本低、简单、高效、灵活且便于携带,适合在现场大批量的样品的快速筛选。本实验采用戊二醛法合成金刚烷胺完全抗原,经透析、鉴定后免疫Balb/c小鼠,利用单克隆抗体技术,酶联免疫吸附试验(ELISA)和小鼠腹水制备等技术,制备出针对金刚烷胺的特异性强、效价高的单克隆抗体,并在此基础上建立检测金刚烷胺的间接竞争ELISA法。1.AMT完全抗原的合成与鉴定AMT属于三环胺类,是饱和三环癸烷的氨基衍生物。戊二醛是同型双功能交联剂,其上的两个醛基可以分别与半抗原和载体蛋白上的伯氨基形成Schiff氏碱,将两分子以五碳链的桥连接起来,合成完全抗原。完全抗原经紫外光谱扫描法鉴定,初步判定偶联成功。最终完全抗原经免疫、细胞融合及ELISA测定,表明两种抗原的制备是成功的。利用BCA法测得AMT-BSA和AMT-OVA的蛋白浓度分别为3.2 mg/mL和3.4 mg/mL。2.AMT单抗制备本实验采取常规免疫方案免疫雌性Balb/c小鼠,用人工合成的AMT-BSA作为免疫原,以AMT-OVA为包被原。五次免疫后第7 d小鼠断尾采血,测血清效价及抑制情况,检测得血清效价达16000以上。利用细胞融合技术、ELISA筛选方法和有限稀释法进行亚克隆获得2株可持续分泌针对AMT抗体的杂交瘤细胞株,分别命名为1D11、2F9。采用小鼠体内诱生法制备抗AMT单克隆抗体的腹水,其蛋白浓度分别为20.7mg/mL和25.3mg/mL;抗体效价分别为32000和128000。采用HiTrap Protein G HP纯化柱纯化腹水,纯化后经SDS-PAGE鉴定杂蛋白明显减少。交叉实验结果表明,该抗体与金刚烷、利巴韦林、吗啉胍、阿莫西林、头孢噻呋几乎没有交叉反应,与盐酸金刚乙胺的交叉反应率为15%,而对AMT的抑制率为100%。3.ELISA检测方法的建立利用纯化后的2F9单克隆抗体建立检测AMT残留的间接竞争ELISA方法。包被原最佳稀释浓度为1:4000,抗体的最佳工作浓度为1:64000,AMT浓度在10～500ng/mL时,标准曲线线性良好,线性方程为y=0.3785x-0.2179(R2=0.9925),IC50为 77.83ng/mL,LOD为 5.98 ng/mL,LOQ 为 54.78 ng/mL。4.鸡肉AMT添加回收实验以鸡肉进行添加AMT回收实验,鸡肉样品经过前处理后用于CiELISA测定。实验结果表明,鸡肉样品处理液稀释16倍以上时样品的基质干扰基本消失。在范围10～1000ng/mL内,曲线标准方程式为 y=0.3377x-0.1658,相关系数 R2=0.9901,IC50 为 92.67ng/mL,LOD为 6.74ng/mL,LOQ 为 132.82ng/mL。用 10、500 和 1000ng/mL 的 AMT 标准液做添加回收实验,经检测鸡肉样品添加回收率在83.4%～102.3%之间。
[Abstract]:Amantadine (AMT) belongs to tricyclic amines. It is the earliest antiviral drug used in the treatment of influenza. It is mainly used for the prevention and treatment of avian influenza and transmissible gastroenteritis in livestock and poultry breeding. However, the side effects of mental disorders and the increasing drug resistance may cause more harm to consumers of animal products. Therefore, the use of amantadine and amantadine and other antiviral drugs in livestock and poultry farming has been prohibited by the announcement No. 560 of the Ministry of Agriculture of China, and the use of such drugs in livestock and poultry farming has been prohibited by FAD of the United States. Chromatography, liquid chromatography-tandem mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, hydrophilic interaction chromatography-tandem mass spectrometry and potentiometric titration, etc. However, traditional instrumental detection methods require not only expensive instruments and equipment, but also professional technicians to operate complex sample processing processes, which are caused by these disadvantages. ELISA is based on the specific binding of antigen and antibody. It has high selectivity and sensitivity. The method is low cost, simple, efficient, flexible and easy to carry. It is suitable for rapid screening of large quantities of samples in the field. Amantadine was synthesized by glutaraldehyde method. Balb/c mice were immunized after dialysis and identification. Monoclonal antibody technology, enzyme-linked immunosorbent assay (ELISA) and mouse ascites preparation were used to prepare monoclonal antibodies against amantadine with high specificity and titer. On this basis, an indirect competitive ELISA method for the detection of amantadine was established. 1. Glutaraldehyde is a bifunctional crosslinking agent. Its two aldehyde groups can form Schiff bases with primary amino groups on hapten and carrier proteins respectively. The two molecules are bridged by a five-carbon chain to synthesize a complete antigen. The complete antigen is scanned by ultraviolet spectrum. The complete antigen was successfully prepared by immunization, cell fusion and ELISA. The protein concentrations of AMT-BSA and AMT-OVA were 3.2 mg/ml and 3.4 mg/ml. AMT-BSA was synthesized as immunogen and AMT-OVA was used as coating agent. On the 7th day after five immunizations, the serum titer and inhibition were measured. The serum titer was more than 16 000. Two hybridoma cell lines secreting antibodies against AMT were obtained by cell fusion, ELISA screening and limited dilution. Ascites containing anti-AMT monoclonal antibodies were prepared by induction in vivo in mice with protein concentrations of 20.7 mg/mL and 25.3 mg/mL, and antibody titers of 32 000 and 128000, respectively. Ascites were purified by HiTrap Protein G-HP column, and the impurity proteins identified by SDS-PAGE were significantly reduced. There were almost no cross reactions with amantadine, ribavirin, morpholine guanidine, amoxicillin and ceftiofur. The cross reaction rate with amantadine hydrochloride was 15% and the inhibition rate against AMT was 100%. 3. Establishment of an indirect competitive ELISA method for detecting AMT residues using purified 2F9 monoclonal antibody. The linear equation was y=0.3785x-0.2179 (R2=0.9925), IC50 was 77.83ng/mL, LOD was 5.98 ng/mL, LOQ was 54.78 ng/mL. In the range of 10-1000ng/mL, the standard equation of the curve is y=0.3377x-0.1658, the correlation coefficient R2=0.9901, the IC50 is 92.67ng/mL, the LOD is 6.74ng/mL, the LOQ is 132.82ng/mL, and the AMT is 10,500 and 1000ng/mL. The recovery rate of chicken samples was between 83.4% and 102.3%.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.84

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