發(fā)布時(shí)間：2018-01-24 02:17

  本文關(guān)鍵詞： 卡那霉素 單克隆抗體 ELISA 納米金顆粒 乳膠微球　出處：《江蘇大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：卡那霉素是一種氨基糖苷類(lèi)抗生素,因其具有良好的抑制細(xì)菌生長(zhǎng)等作用而被廣泛應(yīng)用于醫(yī)藥、農(nóng)業(yè)、畜牧和水產(chǎn)養(yǎng)殖業(yè)�？敲顾氐牟缓侠硎褂�,一方面會(huì)引起卡那霉素在動(dòng)物體內(nèi)的大量殘留,隨著食物鏈的傳遞作用,最終進(jìn)入人體,危害人體健康;另一方面未被吸收利用的卡那霉素會(huì)以原型或其代謝產(chǎn)物的形式排放到自然水體或土壤中,導(dǎo)致嚴(yán)重的環(huán)境污染。因此,建立靈敏度高、特異性強(qiáng)、穩(wěn)定性好、簡(jiǎn)單快捷的分析方法來(lái)檢測(cè)食品和環(huán)境中的卡那霉素含量很有必要。本研究制備了高特異性的卡那霉素單克隆抗體,結(jié)合納米金顆粒建立了卡那霉素直接競(jìng)爭(zhēng)ELISA分析方法,并嘗試建立基于乳膠微球的免疫新方法。本文采用EDC法成功制備了免疫原(Kana-BSA)和包被原(Kana-OVA)。經(jīng)動(dòng)物免疫步驟免疫小鼠后,有3只小鼠體內(nèi)產(chǎn)生的卡那霉素抗體達(dá)到實(shí)驗(yàn)要求,從中挑選出一只血清效價(jià)高且抑制效果好的小鼠,獲取它的脾細(xì)胞與SP2/0細(xì)胞進(jìn)行細(xì)胞融合。融合后經(jīng)篩選,共得到10株能夠穩(wěn)定分泌卡那霉素單克隆抗體的細(xì)胞株。對(duì)10株雜交瘤細(xì)胞株分泌的單抗進(jìn)行類(lèi)與亞型的鑒定,結(jié)果顯示這些單克隆抗體均為IgG1型且輕鏈都為?型。后續(xù)實(shí)驗(yàn)選取A10E5雜交瘤細(xì)胞株分泌的單抗進(jìn)行卡那霉素免疫檢測(cè)方法學(xué)研究。制得Kana-HRP,建立了基于HRP信號(hào)分子的卡那霉素直接競(jìng)爭(zhēng)ELISA方法,此方法的IC50為2.15 ng/mL,LOD為0.13 ng/m L,線性區(qū)間在0.40~9.67 ng/m L;合成Au-HRP-Kana,建立了基于Au-HRP雙標(biāo)記信號(hào)放大的ELISA方法,該方法的IC50為1.05 ng/mL,LOD為0.16 ng/mL,線性區(qū)間為0.31~2.8 ng/m L。A10E5單克隆抗體與妥布霉素交叉反應(yīng)率為99.07%,與其他結(jié)構(gòu)類(lèi)似物的交叉反應(yīng)率均在0.3%以下。實(shí)驗(yàn)測(cè)得加標(biāo)回收率在88.6%~132.8%,變異系數(shù)小于15.8%。對(duì)環(huán)境樣品中卡那霉素殘留情況進(jìn)行分析測(cè)定,結(jié)果發(fā)現(xiàn)江蘇大學(xué)環(huán)境水樣中卡那霉素含量在0.45~2.78 ng/m L,牛奶樣品中卡那霉素的殘留量為0.35~0.55 ng/m L,而土壤中卡那霉素的含量未檢出。實(shí)驗(yàn)采用EDC/NHS法成功合成了乳膠微球-PEG-GAM,建立了基于乳膠微球的免疫新方法,討論了乳膠微球的非特異性結(jié)合情況,并對(duì)實(shí)驗(yàn)條件進(jìn)行了初步的優(yōu)化�；谌槟z微球的離心定量法,其IC50為4.66 ng/m L,LOD為0.25 ng/m L,線性區(qū)間在0.73~19.07ng/mL�；谌槟z微球的過(guò)濾定性法,其最低檢測(cè)限為1 ng/m L,實(shí)驗(yàn)表明可將該方法初步應(yīng)用于對(duì)卡那霉素的定性分析。最后比較了四種檢測(cè)卡那霉素的分析方法,直接競(jìng)爭(zhēng)ELISA方法靈敏度較高,可應(yīng)用于實(shí)際環(huán)境樣品中卡那霉素的分析檢測(cè)。而基于乳膠微球的免疫分析新方法檢測(cè)靈敏度雖沒(méi)有ELISA方法高,但方法簡(jiǎn)單、便捷,可應(yīng)用于對(duì)實(shí)際樣品的初篩。
[Abstract]:Kanamycin is an aminoglycoside antibiotic, which is widely used in medicine, agriculture, animal husbandry and aquaculture. On the one hand, it will cause a large number of kanamycin residues in the animal body, along with the transmission of the food chain, eventually into the human body, harmful to human health; On the other hand, the unabsorbed kanamycin can be discharged into natural water or soil in the form of prototype or its metabolites, resulting in serious environmental pollution. Therefore, the establishment of high sensitivity, specificity and stability. It is necessary to detect kanamycin in food and environment by a simple and rapid analytical method. In this study, a highly specific monoclonal antibody against kanamycin was prepared. A direct competitive ELISA analysis method for kanamycin was established in combination with gold nanoparticles. A new immunization method based on latex microspheres was established. In this paper, the immunogen Kana-BSA and the coated proto-Kana-OVA were successfully prepared by EDC method. The mice were immunized by animal immunization. Three mice produced kanamycin antibody to meet the experimental requirements, and a mouse with high serum titer and good inhibitory effect was selected. The spleen cells were fused with SP2/0 cells. A total of 10 cell lines were obtained to stably secrete monoclonal antibodies against kanamycin. The monoclonal antibodies secreted by 10 hybridoma cell lines were identified by type and subtype identification. The results showed that these monoclonal antibodies were IgG1 type and light chain. Kana-HRP was prepared by using monoclonal antibody secreted by A10E5 hybridoma cell line for kanamycin immunoassay. A ELISA method for direct competition of kanamycin based on HRP signaling molecules was established. The IC50 of this method was 2.15 ng / mLL = 0.13 ng/mL. The linear range is 0. 40 ~ 9. 67 ng/m / L; Au-HRP-Kana was synthesized, and the ELISA method based on Au-HRP double label signal amplification was established. The IC50 of this method was 1.05 ng/mL. LOD was 0.16 ng / mL, and the linear range was 0.31 ~ 2.8 ng/m L. A10E5. The cross reaction rate between monoclonal antibody and tobramycin was 99.07%. The cross reaction rates with other structural analogues were all below 0.3%, and the recoveries were 88.6% and 132.8%, respectively. The coefficient of variation was less than 15.8.The results showed that the content of kanamycin in environmental water sample of Jiangsu University was 0.45 ~ 2.78 ng/m / L. The residual amount of kanamycin in milk samples was 0. 35 and 0. 55 ng/m / L. However, the content of kanamycin in soil was not detected. Latex microspheres PEG-GAM were successfully synthesized by EDC/NHS method, and a new immune method based on latex microspheres was established. The nonspecific binding of latex microspheres was discussed, and the experimental conditions were preliminarily optimized. The IC50 was 4.66 ng/m / L based on the centrifugation quantitative method of latex microspheres. The LOD was 0.25 ng/mL and the linear range was 0.73 ~ 19.07 ng 路mL ~ (-1). The minimum detection limit of the method was 1 ng/mL based on latex microspheres. The results show that this method can be applied to the qualitative analysis of kanamycin. Finally, four analytical methods for the detection of kanamycin are compared, and the sensitivity of the direct competition ELISA method is high. It can be applied to the analysis and detection of kanamycin in practical environmental samples, but the new method based on latex microspheres is not as sensitive as ELISA method, but the method is simple and convenient. It can be used to screen the actual sample.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：X830

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 賈向陽(yáng);尤慧艷;付秀麗;;魚(yú)精蛋白-核酸適配體-金納米技術(shù)快速檢測(cè)牛奶中的卡那霉素[J];色譜;2017年03期

2 徐飛;栗靜雅;周潔;劉茂林;劉義明;王建芬;丁雙陽(yáng);李秀波;;可視化凝膠酶聯(lián)免疫吸附分析法檢測(cè)牛奶中慶大霉素和卡那霉素[J];分析化學(xué);2015年06期

3 宋娟;王榕妹;王悅秋;唐雨榕;鄧安平;;半抗原的設(shè)計(jì)、修飾及人工抗原的制備[J];分析化學(xué);2010年08期

4 李平;鄧省亮;于洪俠;楊曙明;;半抗原特異性抗體的篩選及親和力成熟[J];中國(guó)生物工程雜志;2010年02期

5 劉麗強(qiáng);華朱鳴;許定花;陳偉;胥傳來(lái);;卡那霉素酶聯(lián)免疫檢測(cè)方法的建立及檢測(cè)條件的優(yōu)化[J];食品科學(xué);2009年18期

6 王志強(qiáng);胡國(guó)媛;李志勇;凌莉;胡科峰;;微生物抑制法快速檢測(cè)鮮奶中多種抗生素殘留[J];中國(guó)食品衛(wèi)生雜志;2008年02期

7 習(xí)玲玲;朱巖;;液相色譜-脈沖安培電化學(xué)法測(cè)定硫酸卡那霉素中各組分含量[J];分析化學(xué);2007年05期

8 鄧立新;利用微生物研制新藥[J];福建質(zhì)量信息;2004年10期

9 Schindel L.;劉權(quán);;卡那霉素的副作用[J];山東醫(yī)刊;1965年08期

相關(guān)會(huì)議論文 前1條

1 陳義強(qiáng);肖希龍;李德發(fā);;豬組織中卡那霉素殘留膠體金免疫層析、ELISA及HPLC檢測(cè)方法研究[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)2009學(xué)術(shù)年會(huì)論文集（上冊(cè)）[C];2009年

相關(guān)博士學(xué)位論文 前1條

1 王紅濤;基于分子印跡修飾電極的四環(huán)素選擇性檢測(cè)方法和儀器研究[D];大連理工大學(xué);2011年

相關(guān)碩士學(xué)位論文 前3條

1 韋薇;基于碳納米管的慶大霉素免疫分析方法研究[D];江蘇大學(xué);2016年

2 羅繼寶;適配體傳感器在卡那霉素檢測(cè)中的應(yīng)用[D];江南大學(xué);2015年

3 栗寧;抗卡那霉素單克隆抗體的制備及鑒定[D];鄭州大學(xué);2014年

，

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