本文選題：玉米赤霉烯酮　切入點：單克隆抗體　出處：《上海交通大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：玉米赤霉烯酮(zearalenone,ZEN)是由鐮刀菌屬真菌產(chǎn)生的次級代謝產(chǎn)物,為具有雌性激素活性的真菌毒素。ZEN具有很強的生殖毒性、致畸性和免疫抑制毒性等,人或動物長期食用被ZEN污染的食物或飼料,會對機體造成損害。盡管我國目前對飼料、谷物和谷物食品中的ZEN檢測通常采用高效液相色譜、液-質(zhì)聯(lián)用色譜等方法,但這些方法遠(yuǎn)不能滿足不同樣本中ZEN等真菌毒素的不同檢測需求。本文通過研制抗ZEN單克隆抗體,進而基于免疫層析、免疫傳感、化學(xué)發(fā)光、液相芯片等原理與技術(shù),建立了多種具有自主知識產(chǎn)權(quán)的快速、靈敏、高通量的免疫學(xué)檢測方法,為監(jiān)測谷物、谷物食品、飼料、動物源性食品中ZEN及其他真菌毒素的殘留提供技術(shù)支撐。ZEN是一種半抗原,為了制備其完全抗原,首先合成了玉米赤霉烯酮-6-羧甲氧肟,再通過碳二亞胺法與牛血清白蛋白(BSA)或卵清白蛋白(OVA)偶聯(lián),制備出完全抗原BSA-ZEN和OVA-ZEN。以BSA-ZEN為免疫原,免疫BALB/c小鼠后,取免疫小鼠的脾臟與SP2/0細(xì)胞融合。通過3次篩選和亞克隆,最終獲得了4株穩(wěn)定分泌抗ZEN單克隆抗體的雜交瘤細(xì)胞株。選擇其中效價最高的2C9細(xì)胞株,建立了檢測ZEN的競爭ELISA方法。其檢測限(IC10,即10%抑制濃度)為0.051 ng/mL。交叉反應(yīng)結(jié)果表明,該抗體對ZEN結(jié)構(gòu)類似物的交叉反應(yīng)率為16%,與伏馬毒素b1、嘔吐毒素和黃曲霉毒素b1均無交叉反應(yīng)。為了解決大批量樣品的現(xiàn)場、快速初篩需求,本文基于競爭elisa和免疫層析原理,建立了定性和定量免疫膠體金試紙條檢測技術(shù)。分別用粒徑25nm的膠體金標(biāo)記抗zen單克隆抗體和抗伏馬毒素b1(fb1)單克隆抗體,制成膠體金標(biāo)記抗體墊,以硝酸纖維素膜為層析介質(zhì),分別包被zen偶聯(lián)抗原(bsa-zen)和fb1偶聯(lián)抗原(ova-fb1)作為檢測線,包被羊抗鼠二抗作為質(zhì)控線。在對試紙條制備材料、緩沖液組成和ph、添加物種類和濃度、樣品稀釋度、包被抗原濃度以及金標(biāo)抗體濃度等進行優(yōu)化的基礎(chǔ)上,組裝成試紙條,建立了同時檢測谷物中zen和fb1的雙重定性和定量膠體金試紙條分析方法。雙重定性試紙條對標(biāo)準(zhǔn)品中zen和fb1的檢測限分別為6ng/ml和50ng/ml。雙重定量試紙條對zen的檢測區(qū)間為0.94-7.52ng/ml,檢測限為0.35ng/ml;對fb1的檢測區(qū)間為9.34-100.45ng/ml,檢測限為5.23ng/ml。試紙條的檢測方法不但快速,較此前報道的方法大幅提升了檢測靈敏度。為了實現(xiàn)特定樣品中玉米赤霉烯酮的高靈敏檢測,利用堿性磷酸酶可以將無色、無電化學(xué)活性的對硝基苯磷酸轉(zhuǎn)化為黃色、有電化學(xué)活性的對硝基苯酚的原理,將elisa與電化學(xué)檢測技術(shù)結(jié)合,建立定量檢測分析方法。通過對反應(yīng)緩沖液種類、ph、電化學(xué)參數(shù)、包被抗原濃度、抗體濃度等的優(yōu)化,檢測靈敏度可達0.002ng/ml,檢測區(qū)間為0.004-9.5ng/ml。該方法不但拓寬了檢測區(qū)間,還可實現(xiàn)高通量、高靈敏、快速檢測食品等樣本中痕量的真菌毒素�；诨瘜W(xué)發(fā)光和信號放大系統(tǒng)可提高檢測體系的靈敏度,本文在elisa的基礎(chǔ)上,通過生物素-鏈霉親和素系統(tǒng)、納米金系統(tǒng)的逐個加入,建立了三種直接競爭化學(xué)發(fā)光免疫檢測方法。通過對各方法中反應(yīng)試劑濃度、孵育時間、甲醇濃度等的優(yōu)化,發(fā)現(xiàn)隨著信號放大系統(tǒng)的加入,檢測限逐漸提升,基于納米金系統(tǒng)建立的化學(xué)發(fā)光免疫方法檢測限可至0.008 ng/mL。該方法首次將兩種信號放大策略同時使用,并將雙標(biāo)記納米金(double-codified gold nanoparticles)技術(shù)應(yīng)用于農(nóng)業(yè)和食品檢測領(lǐng)域,為樣本中痕量分析物的檢測提供了又一種快速、精準(zhǔn)的技術(shù)。為了高效檢測谷物中易同時出現(xiàn)的多種真菌毒素(ZEN、FB1、嘔吐毒素和黃曲霉毒素B1),本文將四種抗真菌毒素單克隆抗體分別包被在不同編碼的聚苯乙烯微球(49、39、37、19號)表面,并將四種真菌毒素完全抗原分別與生物素偶聯(lián),最后使用鏈霉親和素-藻紅蛋白作為信號報告蛋白,建立了直接競爭多重液相芯片檢測方法。結(jié)果表明,該方法對ZEN、FB1、嘔吐毒素和黃曲霉毒素B1的檢測限分別為0.51、6.0、4.3和0.56 ng/mL。對于加標(biāo)樣品的檢測,回收率達92.3%-115.5%,與用LC-MS/MS的檢測結(jié)果具有顯著相關(guān)性,表明該方法具有很好的應(yīng)用前景。本論文利用制備的單克隆抗體,分別建立了競爭ELISA法、雙重定性膠體金免疫試紙條檢測法、雙重定量膠體金免疫試紙條檢測法、電化學(xué)免疫法、化學(xué)發(fā)光免疫法和液相芯片多重檢測法,在確保檢測方法特異的前提下,優(yōu)化了檢測過程中的核心參數(shù),拓展了檢測思路,針對不同的檢測樣本,可分別滿足便捷、低成本、高靈敏和高通量等多種檢測需求,為真菌毒素等小分子物質(zhì)的檢測構(gòu)建了一個實用的技術(shù)平臺。
[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.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前1條

1 宋巍巍;丁明星;張挪威;劉海峰;徐明剛;劉國艷;柴春彥;;伏安免疫法檢測牛奶中氯霉素殘留[J];分析化學(xué);2007年12期

相關(guān)碩士學(xué)位論文 前1條

1 唐寧;玉米赤霉烯酮單抗的制備及初步應(yīng)用[D];揚州大學(xué);2009年

，

本文編號：1613917