花状纳米金及抗独特型纳米抗体在真菌毒素免疫学检测中的应用研究

发布时间：2018-01-09 01:19

本文关键词：花状纳米金及抗独特型纳米抗体在真菌毒素免疫学检测中的应用研究　出处：《南昌大学》2017年博士论文　论文类型：学位论文

【摘要】：真菌毒素为产毒真菌产生的一类次级代谢产物,广泛存在于自然界,可经由污染的农产品和食品对人畜的健康产生严重危害,严重时可导致癌症甚至丧命,其中尤以黄曲霉毒素B1(Aflatoxin B1,AFB!)和赭曲霉毒素A(Ochratoxin,OTA)毒性较大,因此建立快速、灵敏且特异性的检测方法显得尤为重要。在检测方法中,免疫学检测方法因具有灵敏度高、特异性强且简单方便等优点是目前常用的真菌毒素检测方法,但随着我国农产品及食品进出口贸易的增加,人们对其质量和安全的要求愈来愈高,普通的免疫学检测方法已经难以满足人们对真菌毒素高灵敏性和特异性的要求,因此建立高灵敏和高特异性的真菌毒素免疫学检测方法势在必行。本论文以建立高灵敏性和特异性的免疫学分析方法为目的,从检测信号及免疫学识别元件两个方面对AFB1和OTA进行新型分析方法的研究。首先合成了具有高光学性质及稳定性的花状纳米金(Gold nanoflowers,AuNFs),以其作为新型标记探针,建立了AFB1高灵敏的免疫层析检测技术;同时,从羊驼天然纳米抗体文库中淘选得到OTA抗独特型纳米抗体(anti-idiotypic nanobody,AId-Nb),以其取代传统的化学合成抗原,建立了高灵敏的环境友好型OTA分析方法。主要研究内容包括:1以对苯二酚作为还原剂,通过种子介导生长法,合成高质量的AuNSs和AuNFs。通过改变种子、氯金酸及对苯二酚的添加量,对AuNSs和AuNFs的合成条件进行初步探索,并通过透射电子显微镜、紫外-可见光-近红外分光光度仪和Zeta电位等表征手段对纳米金的形貌、光学性质及稳定性进行表征。结果显示,种子的添加量为影响粒径大小的主要因素,即随着种子添加量的减少,纳米金的粒径显著增大,并且表面粗糙程度增加,可逐渐形成AuNFs,当种子的加入体积≤500μL时生成AuNFs;反应速率为影响形貌的主要因素,即降低氯金酸添加量(≤150μL)或增加对苯二酚添加量(3000μL≤V≤4000μL)均可生成AuNFs。75nm AuNFs的吸光度是75nm AuNSs的吸光度的1.5倍,表明相同粒径的AuNFs不仅具有更强的光学特性,而且稳定。2以AuNFs作为新型标记探针,建立AFBi的免疫层析试纸条定量检测方法。通过抗AFB1单克隆抗体与AuNFs偶联,以检测线(T线)吸光值与质控线(C线)吸光值的比值(ODT/ODC)进行定量。对大米样品进行检测,并与传统ELISA方法进行结果对比。结果显示,当AFBi浓度在0.5～25 pg/mL范围内,具有较好的线性,且50%抑制浓度(IC50)达到4.17pg/mL(n=5),最低检测限为0.32pg/mL,较传统的AuNSs试纸条的灵敏度提高了 10倍。批内及批间变异系数均小于15%。采用该试纸条和商业化ELISA试剂盒分别分析50份阴性加标大米样品,两者的结果具有较好的线性相关性(R2=0.93)。以AuNFs作为标记探针建立免疫层析检测方法,具有较好的灵敏度,且具有较广阔的商业化应用前景。3以抗OTA单克隆抗体为靶分子,采用“1轮酸洗脱+3轮竞争洗脱”的策略,从羊驼天然纳米抗体噬菌体文库中亲和淘选与之结合的AId-Nb噬菌体,并建立实时荧光定量免疫PCR(Q-IPCR)。经淘选得到一株OTA-AId-Nb噬菌体克隆,并基于该噬菌体建立了 ELISA标准曲线,IC50为300pg/mL,灵敏度较基于传统化学合成抗原的ELISA提高了 8.83倍。进一步构建Q-IPCR,其最低检测限为4.17 pg/mL,比以噬菌体构建的ELISA方法灵敏度提高了 9倍。实际谷物中的Q-IPCR检测结果与商业化ELISA试剂盒检测结果具有较好的相关性。基于AId-Nb的环境友好型的Q-IPCR检测方法为小分子有毒有害污染物的检测提供了新的方法。4以OTA-AId-Nb噬菌体特异性基因构建重组表达载体并将其转入E.coli Rosetta(DE3)中表达。以OTA-AId-Nb取代化学合成抗原,建立了环境友好型ELISA及免疫层析试纸条法。将OTA-AId-Nb作为包被抗原建立ELISA,线性范围为0.08-1.0 ng/mL,IC50为0.25 ng/mL,灵敏度较以化学合成抗原建立的ELISA提高了 10倍。与其它四种真菌毒素(DON、AFBi、FB1、ZEN)无交叉反应。以此OTA-AId-Nb取代传统化学合成抗原,以AuNFs作为标记探针,建立免疫层析检测方法,其cut off值为4 ng/mL,可实现环境友好型现场快速检测。
[Abstract]:A class of secondary metabolites for toxigenic fungi produce mycotoxins, widely exists in the nature, cause serious harm through pollution of agricultural products and food for human and animal health, serious when can cause cancer or even killed, especially in aflatoxin B1 (Aflatoxin B1, AFB!) and ochratoxin A (Ochratoxin OTA), toxicity, so establishing a rapid, sensitive and specific detection method is particularly important. In the detection method, immunological detection methods with high sensitivity, specificity and simple and convenient is a mycotoxin detection method used commonly, but with the increase of China's agricultural products and food import and export trade. The quality and safety of the increasingly high demand, the general immunological detection methods have been difficult to meet the demand of mycotoxins in high sensitivity and specificity, so the establishment of high sensitivity and high specificity It is imperative to mycotoxin immunological detection methods of this paper. For the purpose of establishing immunological analysis method with high sensitivity and specificity, from the two aspects of signal detection and immunological recognition element model analysis method of AFB1 and OTA. The first synthesis of flower like nano gold high optical properties and stability of the Gold (nanoflowers AuNFs), as the new probe labeled an immune chromatography AFB1 high sensitive detection technology; at the same time, from the alpaca natural nano antibody library binding in OTA anti idiotypic antibody (anti-idiotypic nanobody, AId-Nb nano), to replace the traditional chemical synthesis of antigen, the establishment of environmental friendly high sensitive OTA analysis methods. The main research contents include: 1 using hydroquinone as a reducing agent, through seed mediated growth method, synthesis of high quality AuNSs and AuNFs. through the change of seed, gold chloride Adding acid and hydroquinone, explores the synthesis conditions of AuNSs and AuNFs, and by transmission electron microscopy, morphology of gold nanoparticles of ultraviolet visible near infrared spectrophotometer and Zeta potential characterization, optical properties and stability were investigated. The results showed that the adding amount of seeds as the main factors affecting the particle size is decreased with seed addition amount, nano gold particle size increased significantly, and the increase in surface roughness, can be gradually formed when adding AuNFs, seed volume less than or equal to 500 mu L generated AuNFs; the reaction rate for the main factors affecting the morphology, namely reducing chloroauric acid added amount (less than or equal to 150 mu L) or increase the amount of hydroquinone (3000 L = V = 4000 L) can generate AuNFs.75nm AuNFs absorbance is 1.5 times the absorbance of 75Nm AuNSs, showed that the optical properties of the same size AuNFs not only has stronger, and Stable.2 to AuNFs as a new probe labeled immunochromatographic quantitative detection method of the AFBi is established. Through the coupling of anti AFB1 monoclonal antibody and AuNFs to detect line (T line) absorption value and control line (C line) absorption ratio of light value (ODT/ODC) was used for quantitative analysis. To detect the rice samples, and the results were compared with the traditional ELISA method. The results showed that when the concentration of AFBi in 0.5 ~ 25 pg/mL range, good linearity, and the 50% inhibitory concentration (IC50) to 4.17pg/mL (n=5), the lowest detection limit was 0.32pg/mL, compared with the traditional AuNSs test strip sensitivity was 10 times higher than in batch and. Interassay coefficient of variation was less than 15%. by using the test strip and commercial ELISA kit respectively analyzed 50 negative samples spiked rice samples, the result is a good linear correlation (R2=0.93). Using AuNFs as the labeled probe to establish immune chromatography detection method, has better The sensitivity, and has a broad application prospect in commercialization of.3 anti OTA monoclonal antibody as a target molecule, the 1 round +3 round of competition acid elution elution "strategy, from the alpaca natural nano antibody phage library affinity with AId-Nb phage panning, and establish a real-time fluorescence quantitative immuno PCR (Q-IPCR). Strains of OTA-AId-Nb phage clones obtained by phage panning, and based on the established standard curve of ELISA, IC50 for 300pg/mL, the sensitivity is based on the traditional chemical synthesis of antigen ELISA increased 8.83 times. Further construction of Q-IPCR, the lowest detection limit was 4.17 pg/mL, than the ELISA method to construct the phage sensitivity was 9 times higher than Q-IPCR detection. The actual results in grain and commercial ELISA kit test results have good correlation. Environment friendly AId-Nb Q-IPCR detection method based on small molecules for toxic pollutants The test provides a new method of.4 expression vector and transformed into E.coli Rosetta OTA-AId-Nb to construct the recombinant phage specific gene (DE3) expression. To replace the chemical synthesis of antigen to OTA-AId-Nb, the establishment of environmental friendly ELISA and immunochromatographic method. OTA-AId-Nb was used as coating antigen to establish ELISA, linear range is 0.08-1.0 ng/mL, IC50 0.25 ng/mL, the sensitivity is based on the chemical synthesis of the antigen ELISA increased 10 times. And the other four kinds of mycotoxins (DON, AFBi, FB1, ZEN). No cross reaction to OTA-AId-Nb to replace the traditional chemical synthetic antigen, using AuNFs as a probe labeled immunochromatographic method was developed by the cut, off value 4 ng/mL, which can realize rapid detection of environment friendly site.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS207.5;TB383.1

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