本文關(guān)鍵詞： 利巴韋林 金剛烷胺 氫氯噻嗪 ELISA 金標(biāo)試紙條　出處：《江南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來食品的安全問題引起人們的廣泛關(guān)注,快捷,靈敏的免疫檢測(cè)技術(shù)發(fā)展迅猛。本論文從利巴韋林,金剛烷胺和氫氯噻嗪三種常見的違禁藥物出發(fā),首次制備三種藥物的單克隆抗體,并建立了ELISA和膠體金免疫試紙條的檢測(cè)方法,并且能用于實(shí)際樣品的檢測(cè)。通過分析利巴韋林、金剛烷胺和氫氯噻嗪的分子結(jié)構(gòu)特點(diǎn),設(shè)計(jì)不同的半抗原合成方案,分別與琥珀酸酐和6-溴已酸乙酯相連得到合適的半抗原。合成的利巴韋林、金剛烷胺和氫氯噻嗪的半抗原經(jīng)LC-MS鑒定,半抗原均合成成功。在半抗原的基礎(chǔ)上合成三種藥物的完全抗原經(jīng)蛋白質(zhì)凝膠電泳和紫外分光光度法表征合成成功。將合成成功的免疫原免疫BALB/c小鼠,通過4~5次免疫后,選擇高效價(jià)和對(duì)目標(biāo)物抑制效果最好的的小鼠進(jìn)行細(xì)胞融合得到相應(yīng)的單克隆抗體。三種細(xì)胞株的編號(hào)分別為:3E7(RBV)、6H1(AM)和2C3(HCTZ),三種抗體的亞型均是IgG1。通過對(duì)標(biāo)準(zhǔn)品稀釋液中NaCl、pH值和有機(jī)溶劑的含量進(jìn)行優(yōu)化,得到最佳ic-ELISA反應(yīng)條件,從而建立相應(yīng)的ic-ELISA方法。鹽離子濃度為0.8%,不含乙腈,pH值為7.4的最優(yōu)ic-ELISA條件下,利巴韋林抗體的IC50為6.47 ng/m L,線性范圍為1.75~23.95ng/m L。在鹽離子濃度0.8%,不含乙腈,pH值為8.2的最佳ic-ELISA條件下,金剛烷胺抗體的IC50為1.92 ng/m L,線性范圍為0.62~5.89 ng/m L,對(duì)金剛乙胺的交叉反應(yīng)率為9%,IC50為21.30 ng/mL。在乙腈含量為5%的條件下對(duì)利巴韋林抗體和金剛烷胺的IC50均影響不大,可用實(shí)際樣品的檢測(cè)。在鹽離子濃度0.8%,甲醇濃度為5%,pH值為7.4的最優(yōu)ic-ELISA條件下,氫氯噻嗪抗體的線性范圍為0.02~0.36 ng/mL,IC50為0.10ng/m L。建立的ic-ELISA方法用于雞肉和膠囊中的實(shí)際添加檢測(cè),利巴韋林的添加回收率在82.1%~112.3%之間,變異系數(shù)在1.1%~8.4%之間。金剛烷胺的添加回收率在90.3%~116.1%之間,變異系數(shù)在1.0%~10.5%之間。氫氯噻嗪的添加回收率在89.8%~110.2%之間,變異系數(shù)在5.0%~10.6%之間。建立了利巴韋林和金剛烷胺的膠體金試紙條檢測(cè)方法,在雞肉樣品中的消線值分別為50 ng/g和5 ng/g。建立的檢測(cè)方法能用于非法添加利巴韋林、金剛烷胺和氫氯噻嗪的快速檢測(cè)。
[Abstract]:In recent years, food safety issues have attracted wide attention, and rapid and sensitive immunoassay techniques have been developed rapidly. In this paper, ribavirin, amantadine and hydrochlorothiazide are three common banned drugs. The monoclonal antibodies of three drugs were prepared for the first time, and the detection methods of ELISA and colloidal gold immunoassay strip were established, which can be used for the detection of actual samples. The molecular structure characteristics of ribavirin, amantadine and hydrochlorothiazide were analyzed. Different hapten synthesis schemes were designed and linked to succinic anhydride and ethyl 6-bromohexanate respectively to obtain suitable hapten. The hapten of ribavirin, amantadine and hydrochlorothiazide were identified by LC-MS. The complete antigens of the three drugs were synthesized on the basis of hapten and were characterized by protein gel electrophoresis and ultraviolet spectrophotometry. The BALB/c mice were immunized with the synthesized immunogen for 5 times. Monoclonal antibodies were obtained by cell fusion of mice with high titer and best inhibitory effect on the target. The three cell lines were numbered as: 3E7 RBVA1A) and 2C3 HCTZG. The subtypes of the three antibodies were IgG1. The pH value of NaClN and the content of organic solvent were optimized. The optimum ic-ELISA reaction conditions were obtained, and the corresponding ic-ELISA method was established. Under the optimum ic-ELISA conditions, the salt ion concentration was 0.8 and the pH value of acetonitrile was 7.4. The IC50 of ribavirin antibody was 6.47 ng/m / L, and the linear range was 1.75 ~ 23.95ng / m ~ (-1) 路mL ~ (-1). Under the conditions of 0.8 salt ion concentration and no acetonitrile pH 8.2, the best ic-ELISA was obtained. The IC50 of amantadine antibody was 1. 92 ng/mL, the linear range was 0. 62 ~ 5. 89 ng/mL, and the cross reaction rate of amantadine was 9. 30 ng / mL. When the content of acetonitrile was 5%, there was little effect on ribavirin antibody and IC50 of amantadine. The linear range of hydrochlorothiazide antibody is 0.02ng / mL IC50 = 0.10ng / ml 路mL ~ (-1) under the optimal ic-ELISA condition of salt ion concentration 0.8 and methanol pH 7.4. The established ic-ELISA method can be used to detect the actual addition in chicken and capsule, the linear range of hydrochlorothiazide antibody is 0.02ng / mL IC50 0.10ng / mL 路ml ~ (-1). The recovery of ribavirin was between 82.1% and 112.3%, and the coefficient of variation was between 1.1% and 8.4%. The recovery of amantadine was between 90.33% and 116.1%, and the coefficient of variation was between 1.0% and 10.5%. The recovery rate of hydrochlorothiazide was between 89.8% and 110.2%. The coefficient of variation was between 5.0% and 10.6%. A method for the detection of ribavirin and adamantadine in colloidal gold strip was established. The elimination line values of ribavirin and amantadine in chicken samples were 50 ng/g and 5 ng / g, respectively. The established method could be used for illegally adding ribavirin. Rapid detection of amantadine and hydrochlorothiazide.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R927
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本文編號(hào)：1518075