玉米赤霉烯酮单克隆抗体的制备及高通量定性、定量检测技术的研究

发布时间：2018-03-15 02:03

本文选题：玉米赤霉烯酮　切入点：单克隆抗体　出处：《上海交通大学》2014年博士论文　论文类型：学位论文

【摘要】：玉米赤霉烯酮(zearalenone,ZEN)是由镰刀菌属真菌产生的次级代谢产物,为具有雌性激素活性的真菌毒素。ZEN具有很强的生殖毒性、致畸性和免疫抑制毒性等,人或动物长期食用被ZEN污染的食物或饲料,会对机体造成损害。尽管我国目前对饲料、谷物和谷物食品中的ZEN检测通常采用高效液相色谱、液-质联用色谱等方法,但这些方法远不能满足不同样本中ZEN等真菌毒素的不同检测需求。本文通过研制抗ZEN单克隆抗体,进而基于免疫层析、免疫传感、化学发光、液相芯片等原理与技术,建立了多种具有自主知识产权的快速、灵敏、高通量的免疫学检测方法,为监测谷物、谷物食品、饲料、动物源性食品中ZEN及其他真菌毒素的残留提供技术支撑。ZEN是一种半抗原,为了制备其完全抗原,首先合成了玉米赤霉烯酮-6-羧甲氧肟,再通过碳二亚胺法与牛血清白蛋白(BSA)或卵清白蛋白(OVA)偶联,制备出完全抗原BSA-ZEN和OVA-ZEN。以BSA-ZEN为免疫原,免疫BALB/c小鼠后,取免疫小鼠的脾脏与SP2/0细胞融合。通过3次筛选和亚克隆,最终获得了4株稳定分泌抗ZEN单克隆抗体的杂交瘤细胞株。选择其中效价最高的2C9细胞株,建立了检测ZEN的竞争ELISA方法。其检测限(IC10,即10%抑制浓度)为0.051 ng/mL。交叉反应结果表明,该抗体对ZEN结构类似物的交叉反应率为16%,与伏马毒素b1、呕吐毒素和黄曲霉毒素b1均无交叉反应。为了解决大批量样品的现场、快速初筛需求,本文基于竞争elisa和免疫层析原理,建立了定性和定量免疫胶体金试纸条检测技术。分别用粒径25nm的胶体金标记抗zen单克隆抗体和抗伏马毒素b1(fb1)单克隆抗体,制成胶体金标记抗体垫,以硝酸纤维素膜为层析介质,分别包被zen偶联抗原(bsa-zen)和fb1偶联抗原(ova-fb1)作为检测线,包被羊抗鼠二抗作为质控线。在对试纸条制备材料、缓冲液组成和ph、添加物种类和浓度、样品稀释度、包被抗原浓度以及金标抗体浓度等进行优化的基础上,组装成试纸条,建立了同时检测谷物中zen和fb1的双重定性和定量胶体金试纸条分析方法。双重定性试纸条对标准品中zen和fb1的检测限分别为6ng/ml和50ng/ml。双重定量试纸条对zen的检测区间为0.94-7.52ng/ml,检测限为0.35ng/ml;对fb1的检测区间为9.34-100.45ng/ml,检测限为5.23ng/ml。试纸条的检测方法不但快速,较此前报道的方法大幅提升了检测灵敏度。为了实现特定样品中玉米赤霉烯酮的高灵敏检测,利用碱性磷酸酶可以将无色、无电化学活性的对硝基苯磷酸转化为黄色、有电化学活性的对硝基苯酚的原理,将elisa与电化学检测技术结合,建立定量检测分析方法。通过对反应缓冲液种类、ph、电化学参数、包被抗原浓度、抗体浓度等的优化,检测灵敏度可达0.002ng/ml,检测区间为0.004-9.5ng/ml。该方法不但拓宽了检测区间,还可实现高通量、高灵敏、快速检测食品等样本中痕量的真菌毒素。基于化学发光和信号放大系统可提高检测体系的灵敏度,本文在elisa的基础上,通过生物素-链霉亲和素系统、纳米金系统的逐个加入,建立了三种直接竞争化学发光免疫检测方法。通过对各方法中反应试剂浓度、孵育时间、甲醇浓度等的优化,发现随着信号放大系统的加入,检测限逐渐提升,基于纳米金系统建立的化学发光免疫方法检测限可至0.008 ng/mL。该方法首次将两种信号放大策略同时使用,并将双标记纳米金(double-codified gold nanoparticles)技术应用于农业和食品检测领域,为样本中痕量分析物的检测提供了又一种快速、精准的技术。为了高效检测谷物中易同时出现的多种真菌毒素(ZEN、FB1、呕吐毒素和黄曲霉毒素B1),本文将四种抗真菌毒素单克隆抗体分别包被在不同编码的聚苯乙烯微球(49、39、37、19号)表面,并将四种真菌毒素完全抗原分别与生物素偶联,最后使用链霉亲和素-藻红蛋白作为信号报告蛋白,建立了直接竞争多重液相芯片检测方法。结果表明,该方法对ZEN、FB1、呕吐毒素和黄曲霉毒素B1的检测限分别为0.51、6.0、4.3和0.56 ng/mL。对于加标样品的检测,回收率达92.3%-115.5%,与用LC-MS/MS的检测结果具有显著相关性,表明该方法具有很好的应用前景。本论文利用制备的单克隆抗体,分别建立了竞争ELISA法、双重定性胶体金免疫试纸条检测法、双重定量胶体金免疫试纸条检测法、电化学免疫法、化学发光免疫法和液相芯片多重检测法,在确保检测方法特异的前提下,优化了检测过程中的核心参数,拓展了检测思路,针对不同的检测样本,可分别满足便捷、低成本、高灵敏和高通量等多种检测需求,为真菌毒素等小分子物质的检测构建了一个实用的技术平台。
[Abstract]:Zearalenone (zearalenone, ZEN) is a genus of fungi secondary metabolites produced by Fusarium, as has estrogen activity of mycotoxin.ZEN has strong reproductive toxicity, teratogenicity and immune suppression toxicity, human or animal ZEN long-term consumption of contaminated food or feed, will cause damage to the body. Although China's current feed, cereals and cereal food ZEN detection in high performance liquid chromatography is usually used, combined with liquid chromatography - mass, but these methods can not meet the ZEN different mycotoxins in the sample of different detection requirements. Through the development of anti ZEN monoclonal antibody, and then based on immunochromatography, immunosensor, chemical light, the principle and technology of liquid chip, established with independent intellectual property rights of the rapid, sensitive and high-throughput immunological detection methods for monitoring the grain, grain, food, feed, animal derived food In ZEN and other mycotoxins residue to provide technical support for.ZEN is a semi antigen, in order to prepare the complete antigen, we synthesized zearalenone -6- carboxymethyl hydroxamic, followed by carbon two imide method with bovine serum albumin (BSA) or ovalbumin (OVA) were prepared by coupling. BSA-ZEN and OVA-ZEN. complete antigen with BSA-ZEN as immunogen to immunize BALB/c mice after immunized mouse spleen cells with SP2/0 fusion. Through the 3 screening and subcloning, obtained 4 strains secreting anti ZEN monoclonal antibody hybridoma cell line. The highest titer of 2C9 cell line, establish competition ELISA method for the detection of ZEN. The detection limit (IC10, namely 10% inhibitory concentration) of 0.051 ng/mL. indicate the cross reaction results, the antibodies cross reacted with the ZEN analogs was 16%, with fumonisin B1, emetic toxin and aflatoxin B1 had no cross reaction. In order to solve large quantities of samples, rapid screening of demand, the competition of ELISA and immune chromatography based on the principle of the establishment of qualitative and quantitative immune colloidal gold test strip detection technology were used. The particle diameter of colloidal gold 25nm labeled anti Zen monoclonal antibody and anti fumonisin B1 (FB1) monoclonal antibody, made of colloidal gold remember the antibody pad, with nitrocellulose membrane as adsorbent, which are coated by Zen antigen (bsa-zen) and FB1 (ova-fb1) as antigen detection line, coated with Sheep anti mouse antibody as control line in two. The strip material preparation, buffer composition and pH, additive and concentration, sample dilution degree of concentration of coating antigen and gold labeled antibody concentration were optimized on the basis of the assembled test strip was developed for the determination of Zen and FB1 in grains both qualitative and quantitative analysis methods. The colloidal gold strip of double strip of standard The detection limit of Zen and FB1 products were 6ng/ml and 50ng/ml. double quantitative strip for Zen detection range of 0.94-7.52ng/ml, the detection limit is 0.35ng/ml; detection interval of FB1 is 9.34-100.45ng/ml, the detection limit of detection method of 5.23ng/ml. strip method is not only fast than previously reported significantly enhance the detection sensitivity of high sensitivity. In order to achieve specific detection corn samples zearalenone, using alkaline phosphatase can be colorless, no electrochemical activity of p-nitrophenyl phosphate into yellow, principle of p-nitrophenol electrochemical activity, combining ELISA with electrochemical detection technology, the establishment of quantitative analysis method. Based on the reaction buffer type, pH. The electrochemical parameters, antigen concentration, optimal antibody concentration, detection sensitivity of 0.002ng/ml detection range of 0.004-9.5ng/ml. the method can not only widen the detection The interval, can achieve high throughput, high sensitivity, rapid detection of trace food samples of mycotoxins. Chemiluminescence and signal amplification system can improve the sensitivity of detection system based on ELISA in this paper on the basis of the biotin streptavidin system, gold nanoparticle system added one by one, set up three kinds of direct competitive chemiluminescence immunoassay method. By means of the method of reagent concentration, incubation time, optimization of methanol concentration, with the added signal amplification system, the detection limit is gradually improving, the establishment of the system of nano gold chemical luminescence immune detection limit to 0.008 ng/mL. for the first time the method of two kinds of signal amplification strategy at the same time, based on the use of the double labeled gold nanoparticles (double-codified gold nanoparticles) technology is applied to agriculture and food detection field, for the detection of trace analytes in the sample provided another A fast, precise technology. In order to efficiently detect a variety of mycotoxins in cereals is easy to appear at the same time (ZEN, FB1, emetic toxin and aflatoxin B1), the four anti toxin monoclonal antibodies were immobilized on polystyrene microspheres with different encoding (49,39,37,19) surface, and four kinds of mycotoxins and complete antigen biotin conjugated, finally using streptavidin phycoerythrin as signal reporter protein detection method was established in direct competition with multiple liquid chip. The results show that the method of ZEN, FB1, the detection limit of vomitoxin and aspergillus toxin B1 were 0.51,6.0,4.3 and 0.56 ng/mL. for detecting samples spiked recovery the rate of 92.3%-115.5% has significant correlation with the results of LC-MS/MS detection showed that the method has good application prospect. The preparation of monoclonal antibody against ELI were established, the competition SA method, dual qualitative immune colloidal gold test strip method, double quantitative colloidal gold Immunostrip assay, electrochemical immunoassay method, multiple detection method of chemiluminescence immunoassay and liquid chip, in the premise to ensure the specific detection method, the key parameters of the detection process optimization, expand the testing ideas for detection different samples were met, convenient, low cost, high sensitivity and high throughput requirements and other detection, to build a practical platform for the detection of mycotoxins and other small molecules.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R392

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