發(fā)布時(shí)間：2018-01-14 12:32

  本文關(guān)鍵詞：桔青霉素單克隆抗體制備及三種真菌毒素蛋白芯片檢測(cè)方法的研究　出處：《河北大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 真菌毒素 單克隆抗體 蛋白芯片 檢測(cè)方法

【摘要】：背景:真菌毒素為真菌產(chǎn)生的次級(jí)代謝產(chǎn)物,具有致癌、致突變、致畸等多種毒性,分布在小麥、玉米、飼料及多種食品中,一旦大量進(jìn)入生態(tài)環(huán)境,將對(duì)動(dòng)物、人及整個(gè)生物環(huán)境產(chǎn)生嚴(yán)重危害,因此倍受全球廣泛關(guān)注。目前真菌毒素的檢測(cè)方法主要有理化方法和免疫檢測(cè)方法,理化方法靈敏度較高,但操作復(fù)雜,儀器昂貴,免疫方法一次只能檢測(cè)一種毒素。因此,高通量的蛋白芯片方法將成為食品中真菌毒素快速檢測(cè)的發(fā)展方向。 方法:本實(shí)驗(yàn)先通過(guò)抗原制備、抗原鑒定、免疫、細(xì)胞融合與篩選等步驟制備桔青霉素單克隆抗體,然后以桔青霉素(citrinin,CIT)、脫氧雪腐鐮刀菌烯醇(Deoxynivalenol,DON)和伏馬菌素B1(fumonisin,B1)為研究對(duì)象,采用間接競(jìng)爭(zhēng)免疫原理,以熒光信號(hào)為檢測(cè)信號(hào),確定CIT、DON和FB1人工抗原的最佳固定濃度、固定方式,抗CIT、抗DON、抗FB1三種單抗和IgG-Cy5二抗的最佳工作濃度,建立單獨(dú)檢測(cè)一種毒素的蛋白質(zhì)芯片檢測(cè)方法。并在此基礎(chǔ)上初步建立同時(shí)檢測(cè)三種真菌毒素的蛋白質(zhì)芯片方法。 結(jié)果:(1)經(jīng)過(guò)三種方法的鑒定,桔青霉素人工抗原偶聯(lián)成功;(2)通過(guò)免疫小鼠制備雜交瘤細(xì)胞株,獲得了一株抗桔青霉素單克隆抗體的細(xì)胞株;(3)建立了桔青霉素間接競(jìng)爭(zhēng)ELISA,其IC50為96.2 ng/ml,檢測(cè)限為10 ng/ml,線性范圍為10-810 ng/ml;(4)對(duì)大麥、玉米、飼料樣品進(jìn)行添加回收實(shí)驗(yàn),回收率分別為99-127.1%、75.4-124.5%、78-108%。(5)確定了CIT、DON和FB1人工抗原固定最佳工作濃度,分別為1:100、1:500、1:250;三種真菌毒素單克隆抗體的最佳工作濃度為1:500、1:400、1:4000;IgG-Cy5二抗的最佳工作濃度為1:1000,最佳人工抗原固定條件是25℃固定16 h,繪制了三種真菌毒素競(jìng)爭(zhēng)抑制曲線,IC50分別為63.7 ng/mL、83.5 ng/mL、54.6 ng/mL,線性范圍分別為:10-810 ng/mL、10-810 ng/mL、1.9-125 ng/mL。(6)繪制了三種真菌毒素同時(shí)檢測(cè)的競(jìng)爭(zhēng)抑制曲線,CIT靈敏度(IC50)為25.7 ng/mL,線性范圍10-810 ng/mL,DON靈敏度(IC50)為58.5 ng/mL,線性范圍10-810 ng/mL, FB1靈敏度(IC50)為24.9 ng/mL,線性范圍1.9-125 ng/mL。 結(jié)論:通過(guò)制備抗桔青霉素單克隆抗體建立間接競(jìng)爭(zhēng)ELISA,經(jīng)添加回收試驗(yàn)初步驗(yàn)證該ELISA檢測(cè)方法可用于實(shí)際樣品的檢測(cè);根據(jù)我國(guó)規(guī)定的限量標(biāo)準(zhǔn),初步建立的同時(shí)檢測(cè)三種真菌毒素的蛋白芯片檢測(cè)方法滿足我國(guó)限量需求。
[Abstract]:Background: mycotoxins are secondary metabolites produced by fungi. They are carcinogenic, mutagenic and teratogenic. They are distributed in wheat, corn, feed and many kinds of food. It will cause serious harm to animals, human beings and the whole biological environment, so it has attracted worldwide attention. At present, the detection methods of mycotoxins are mainly physicochemical methods and immunoassay methods, and the sensitivity of physicochemical methods is high. But the operation is complicated, the instrument is expensive, the immune method can only detect one toxin at a time. Therefore, the high-throughput protein chip method will become the development direction of rapid detection of mycotoxins in food. Methods: the monoclonal antibody against citrinin was prepared by antigen preparation, antigen identification, immunity, cell fusion and screening, and then citrinin cit was used. Deoxynivalenolol (DON) and fumonisinB1 (fumonisinine B1) were used in the study. Using fluorescence signal as detection signal, the best fixed concentration, fixation mode, anti-CITand and anti-#en1# of CITDON and FB1 artificial antigens were determined. The optimum working concentration of three kinds of monoclonal antibodies and IgG-Cy5 second antibodies against FB1. A protein chip method for the detection of a single toxin was established and a protein chip method for the simultaneous detection of three mycotoxins was established. Results 1) after three methods of identification, the conjugation of citrinin artificial antigen was successful. (2) the hybridoma cell line was prepared by immunizing mice, and a cell line with monoclonal antibody against citrinin was obtained. (3) the indirect competition of citrinin was established, the IC50 was 96. 2 ng / ml, the detection limit was 10 ng / ml, and the linear range was 10-810 ng / ml; The recovery rates of barley, corn and feed samples were 99-127.1 and 75.4-124.5and 78-108.The recovery rate was 75.4-124.5. the CIT was determined. The optimal working concentration of DON and FB1 artificial antigen fixation was 1: 100 1: 500 and 1: 250, respectively. The optimal working concentration of monoclonal antibodies against three mycotoxins was 1: 500, 1: 400, 1: 4 000; The optimal working concentration of the second IgG-Cy5 antibody was 1: 1000, and the best condition of artificial antigen fixation was 25 鈩，

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