T-2毒素免疫学快速检测方法的建立

发布时间：2018-03-21 07:30

本文选题：T-2毒素　切入点：人工抗原　出处：《河南科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：T-2毒素是一种毒性较强的霉菌毒素,可通过污染农作物及饲料导致人和动物中毒。目前,检测T-2毒素最主要的是仪器分析方法。仪器检测方法虽然灵敏度高,但由于技术要求高且不适于现场快速检测,因此限制了其使用范围,由于免疫分析技术具有速度快、操作简单等显著优点,在兽药残留、农药残留、违禁添加物监控检测领域得到广泛应用。本研究拟通过T-2毒素抗原结构分析,制备出人工抗原;并通过动物免疫、细胞融合及阳性杂交瘤筛选制备T-2毒素单克隆抗体;并在此基础上研制出T-2毒素快速检测ELISA试剂盒。本研究的主要内容及结果如下:1.T-2毒素人工抗原的合成与鉴定对T-2毒素化学结构进行分析,针对T-2毒素的化学结构中含有的羟基活性基团,选取牛血清蛋白(BSA)和鸡卵清蛋白(OVA)作为载体蛋白,利用N,N’-羰基二咪唑(CDI)法合成人工抗原T-2-BSA和T-2-OVA,并采用紫外扫描、SDS-PAGE电泳及动物免疫对人工抗原进行了鉴定。结果显示,偶联蛋白的吸收峰与载体蛋白吸收峰没有重叠,但略有偏移,且载体蛋白的迁移速率比偶联后的蛋白要快,由此初步推断人工抗原偶联成功;T-2-BSA所免疫小鼠的多抗血清效价高达1:1×104,半数抑制浓度(IC_(50))为8.32 ng/mL,说明所制备的人工抗原具有良好的免疫原性,且能诱导动物机体产生针对T-2毒素的特异性抗体。2.T-2毒素单克隆抗体的制备及鉴定选取T-2-BSA免疫小鼠中血清效价高、抑制效价好的小鼠作为融合对象。通过细胞融合技术将小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,利用ELISA筛选体系对融合后的细胞进行筛选,选取阳性杂交瘤细胞进行扩大培养亚克隆,最终筛选出1株可以稳定产生抗T-2毒素单克隆抗体的杂交瘤细胞株,命名为4F7,经测定细胞上清效价在1:1×10~3以上,采用体内诱生腹水法制备腹水,采集的腹水效价高达1:6.4×105,且对T-2毒素的半数抑制IC_(50)可以达到2.28ng/mL,该单克隆抗体的亚型为Ig G1型,亲和常数Ka为1.49×1011 L/mol,与其它霉菌毒素交叉反应率均低于0.01%。3.T-2毒素间接竞争ELISA快速检测试剂盒的制备采用间接竞争ELISA棋盘法方法确定包被原包被浓度及抗体工作浓度,通过对反应时间的优化,建立间接竞争ELISA检测方法从而制备出T-2毒素间接竞争ELISA快速检测试剂盒。在包被原浓度为1:4000稀释,抗体工作浓度为1:32000稀释时所制备的T-2毒素快速检测试剂盒的标准曲线的线性回归方程为y=-0.2742x+0.5939,R~2=0.9911,根据方程计算出IC_(50)为2.19 ng/mL,灵敏度可以达到0.129 ng/m L,添加回收率在83.8%~94.3%之间,变异系数小于15%且交叉反应率均小于0.01%,在4°C存放180 d未出现质量问题。
[Abstract]:T-2 toxin is a highly toxic mycotoxin, which can lead to human and animal poisoning by contaminating crops and feed. At present, the most important method for detecting T-2 toxin is instrument analysis. However, due to the high technical requirements and unsuitable for rapid detection in the field, the scope of its application is limited. Due to the remarkable advantages of the immunoassay technology, such as high speed and simple operation, the residues of veterinary drugs and pesticides are found in the veterinary medicine residues, pesticide residues, and so on. In this study, artificial antigens were prepared by analyzing the structure of T-2 toxin antigen, and monoclonal antibodies against T-2 toxin were prepared by animal immunity, cell fusion and positive hybridoma screening. On this basis, the ELISA kit for rapid detection of T-2 toxin was developed. The main contents and results of this study were as follows: 1. The synthesis and identification of T-2 toxin artificial antigen to analyze the chemical structure of T-2 toxin. Bovine serum protein (BSA) and chicken ovalbumin (OVA) were selected as carrier proteins for the hydroxyl groups contained in the chemical structure of T-2 toxin. The artificial antigens T-2-BSA and T-2-OVA were synthesized by the method of NNN-carbonyldiimidazolium (CDI), and the artificial antigens were identified by UV-scanning SDS-PAGE and animal immunity. The results showed that the absorption peaks of conjugated proteins did not overlap with the absorption peaks of carrier proteins, but shifted slightly. The transfer rate of carrier protein was faster than that of coupling protein. It was preliminarily inferred that the polyclonal antibody titer of mice immunized with artificial antigen coupled successfully with T-2-BSA was as high as 1: 1 脳 10 ~ 4 and ICM _ (50) was 8.32 ng / mL, which indicated that the artificial antigen prepared had good immunogenicity. The monoclonal antibody against T-2 toxin was produced in animal body. 2. Preparation and identification of monoclonal antibody against T-2 toxin in mice immunized with T-2-BSA. The spleen cells of mice were fused with SP2/0 myeloma cells by cell fusion technique, and the fused cells were screened by ELISA screening system. A hybridoma cell line, named 4F7, was selected to produce monoclonal antibody against T-2 toxin. The supernatant titer of the cell supernatant was over 1: 1 脳 10 ~ (-3). Ascites were prepared by inducing ascites in vivo. The titer of ascites collected was as high as 1: 6.4 脳 105, and the half inhibition of T-2 toxin was 2.28 ng / mL. the monoclonal antibody subtype was Ig G1. The affinity constant Ka was 1.49 脳 1011 L / mol, and the cross reaction rate with other mycotoxins was lower than 0.01. 3. The preparation of indirect competitive ELISA kit for toxin was determined by indirect competitive ELISA chessboard method. By optimizing the reaction time, an indirect competitive ELISA detection method was established to prepare the T-2 toxin indirect competitive ELISA rapid detection kit, which was diluted at the original concentration of 1: 4000. The linear regression equation of the standard curve of the T-2 toxin rapid detection kit prepared when the working concentration of antibody is 1: 32000 dilution is yap-0.2742x 0.5939 ~ (+) ~ 0.9911. According to the equation, the ICS50 is calculated as 2.19 ng / mL. the sensitivity can reach 0.129 ng / mL, and the recovery rate is between 83.894% and 94.3%. The coefficient of variation was less than 15% and the cross reaction rate was less than 0.01. There was no quality problem at 4 掳C for 180 days.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8

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