【作者】 李涛；

【导师】 刘代成； 李培武；

【作者基本信息】 山东师范大学 ， 食品科学， 2008， 硕士

【摘要】 黄曲霉毒素（Aflatoxins, AFT）是黄曲霉（Aspergillus flavus）和寄生曲霉（A.parasiticus）等产生的一组结构类似的次生代谢产物。其中黄曲霉毒素B1（Aflatoxin B1, AFB₁）是迄今发现的毒性最大、致癌性最强的一种真菌毒素。调查表明,世界范围内AFT污染十分普遍。因此,AFT受到最广泛的重视,各国政府都制定了十分严格的食品中AFT的限量标准。自20世纪六十年代以来,已逐渐探索出了多种AFT的检测方法。这些方法都曾在各个历史时期为检测AFT作出了一定贡献,但也存在着操作复杂、污染大、对仪器设备要求高、假阳性率高、检测时间长等缺陷。随着时代发展,大部分方法已经不能适应当代社会要求的污染小、灵敏度高、检测时间短及操作简单的检测要求。以免疫分析为基础的准确、快速、无污染、智能化检测技术已成为AFB₁快速检测方法研究的热点。其中免疫亲和层析技术由于其对AFT的提取效率高、净化程度高,越来越受研究人员的推崇,该方法是目前公认的最有效的AFB₁净化方法。而高亲和力、高灵敏度、质量稳定的抗体是建立理想检测方法的前提,是应首先解决的问题。本研究的主要内容包括:抗AFB₁单克隆抗体的制备和特性鉴定及其免疫亲和柱的初步研制。目的:通过杂交瘤细胞技术制备抗AFB₁单克隆抗体,并对所制备的单克隆抗体进行特性鉴定和初步应用。方法:（1）以AFB₁-BSA作为抗原免疫BALB/c小鼠;（2）取免疫效果最佳的小鼠脾细胞与骨髓瘤细胞SP2/0进行融合;（3）以AFB₁-OVA作为检测抗原,通过酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）检测杂交瘤细胞的上清进行筛选,保留阳性孔,用有限稀释法进行三次克隆化,得到能稳定分泌单克隆抗体的杂交瘤细胞株;（4）探索大量制备和纯化所得抗体的方法和条件;（5）鉴定对所得单克隆抗体的特性（6）初步应用该单克隆抗体,研制AFB₁免疫亲和柱。结果:（1）得到一株能稳定分泌抗AFB₁单克隆抗体的杂交瘤细胞株5E10。此单克隆抗体的亚类为IgG₂b,亲和常数是4.79×10⁸M^-1,与AFB₂、AFG₁、AFG₂、AFM₁的交叉反应率分别为<26.06%、63.68%、26.06%、39.26%。（2）单克隆抗体的大量制备采用动物体内生产的方法,即以不完全弗氏佐剂（freund’s incomplete adjuvant, FIA）为致敏剂预处理12周龄以上的BALB/c小鼠。一周内腹腔注射杂交瘤细胞1～5×10⁶个/只,10天后便可采集腹水;辛酸硫酸铵法（CA-AS）适用于该单抗的大量纯化。（3）初步制备AFB₁单抗免疫亲和柱,采用荧光分光光度法确定AFB₁单抗免疫亲和柱的使用条件。使用条件为:5mL 0.01M PBS（pH7.4）平衡;上样缓冲液中甲醇浓度为10%;上样后2mL超纯水洗涤;1mL80%甲醇-水洗脱,20mL0.01M PBS（pH7.4）再生。制备的AFB₁单抗免疫亲和柱其抗体浓度与柱容量的关系为y=85.63x-0.043,其中R²=0.9732。制备柱容量为40ng的免疫亲和柱,使用1次后,柱容量下降至原柱容量的43.6%。结论:杂交瘤细胞株5E10能稳定的分泌目的单克隆抗体,利用该抗体初步研制的AFB₁免疫亲和柱的性能仍需进一步优化。更多还原

【Abstract】 Aflatoxin （AFT） is a group of structurally similar secondary metabolites, produced by Aspergillus flavus and A.parasiticu, aflatoxin B1 （AFB₁） is the most serious occurring chemical carcinogens and is one of the most toxic known to a fungal toxin. Investigation shows that aflatoxin contamination is widespread in the world. Therefore, the most widely attention pays to aflatoxin, governments have enacted very stringent standards to limit amount of aflatoxin in food. Since the 1950s, it gradually explored many ways to detect AFB₁, these methods have made some contribution in various historical periods for detecting aflatoxin, however, it also has shortcomings, such as complicated operating, pollution, high equipment requirement, the high false positive rate, long detection time; with the development of the times, it has been unable to meet the requirements of contemporary society that small pollution, high sensitivity, short detection time, simple testing. The accurate, rapid, clean, intelligent detection technology on the basis of immunoassay has become the hot of the rapid test to AFB₁. Because of the high extraction efficiency, high degree of purification, immunoaffinity chromatography technology to aflatoxin is increasingly respected by the research staff, this method is recognized as the most effective purification methods. High affinity, high sensitivity, stable quality antibody is the premise which should be solved firstly to establish the ideal testing method.In this study, monoclonal antibodies （McAb） against aflatoxin B1 was prepared and its characterizations were identified, then we preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.Objective: Prepared the monoclonal antibodies against aflatoxin B1 by hybridoma technique, identify its characterizations and preliminary apply the monoclonal antibodies.Method: （1） BALB/c mice were immunized with AFB₁-BSA; （2） Hybridomas were produced by fusing SP2/0 myeloma cells with spleen cells from the immunized BALB/c which was immune best; （3） Supernatant of the growing hybirdoma was used to screen for the presence of anti-AFB₁-BSA antibody with ELISA. The positive clone was subcloned for three cycles by limiting dilution to select stable secreting hybridoma; （4） Explored the method and conditions to prepare the large number of antibodies and antibodies purification; （5） Identified monoclonal antibodies’characterizations; （6） Preliminary apply the monoclonal antibodies to prepare the immunoaffinity column.Result: （1） One hybirdoma secreting McAb against AFB₁ was produced and its subclass of the McAb was the IgG₂b, its affinity constant was 4.79×10⁸M^-1, and its cross-reaction rates with AFB₂, AFG₁, AFG₂, AFM₁ were <26.06%、63.68%、26.06%、39.26% ; （2） Freund’s Incomplete adjuvant retreated 10 to 12 weeks mice to prepare the mice ascites, then purified by the method of CA-AS; （3） AFB₁ immunoaffinity column’s procedures were 5mL 0.01M PBS （pH7.4） balanced the column, methanol concentration of the sample buffer was 10%, then 2mL ultrapure water wash, 1mL 80% methanol-water elute, 20mL0.01M PBS （pH7.4） regenerate; column capacity and antibodies’concentration relation formula is y=85.63x-0.043, R2=0.9732; the column that the column capacity was 40ng desend to 43.6% after one test.Conclusion: The cell line 5E10 could secret the purpose McAb, AFB₁ immunoaffinity column prepared by the McAb needs to be further optimized.更多还原