【摘要】：金剛烷胺(Amantadine,AMT)屬于三環(huán)胺類,是最早用于治療流感的抗病毒藥。在畜禽養(yǎng)殖中,主要用于治療和預(yù)防雞禽流感,以及豬傳染性胃腸炎的防治,但是其致精神異常的副作用以及日益增強的耐藥性對畜產(chǎn)品消費者可能造成的危害越來越引起關(guān)注。因此,我國農(nóng)業(yè)部560號公告中已明令禁止金剛烷胺和金剛乙胺等抗病毒藥物用于畜禽養(yǎng)殖業(yè),美國FAD也已明確禁止在畜禽養(yǎng)殖中使用此類藥物。檢測金剛烷胺的殘留可采用傳統(tǒng)的儀器檢測方法,主要有液相色譜法、氣相色譜法、液相色譜-串聯(lián)質(zhì)譜法、超高液相色譜-串聯(lián)質(zhì)譜法、親水作用色譜-串聯(lián)質(zhì)譜法以及電位滴定法等。然而,傳統(tǒng)的儀器檢測方法不僅需要昂貴的儀器設(shè)備,復(fù)雜的樣品處理過程還需要專業(yè)的技術(shù)人員進行操作,這些不利因素使這些檢測方法不適用于現(xiàn)場高通量的樣品檢測。ELISA檢測方法基于抗原抗體特異性結(jié)合,具有高度的選擇性和靈敏度,檢測方法成本低、簡單、高效、靈活且便于攜帶,適合在現(xiàn)場大批量的樣品的快速篩選。本實驗采用戊二醛法合成金剛烷胺完全抗原,經(jīng)透析、鑒定后免疫Balb/c小鼠,利用單克隆抗體技術(shù),酶聯(lián)免疫吸附試驗(ELISA)和小鼠腹水制備等技術(shù),制備出針對金剛烷胺的特異性強、效價高的單克隆抗體,并在此基礎(chǔ)上建立檢測金剛烷胺的間接競爭ELISA法。1.AMT完全抗原的合成與鑒定AMT屬于三環(huán)胺類,是飽和三環(huán)癸烷的氨基衍生物。戊二醛是同型雙功能交聯(lián)劑,其上的兩個醛基可以分別與半抗原和載體蛋白上的伯氨基形成Schiff氏堿,將兩分子以五碳鏈的橋連接起來,合成完全抗原。完全抗原經(jīng)紫外光譜掃描法鑒定,初步判定偶聯(lián)成功。最終完全抗原經(jīng)免疫、細胞融合及ELISA測定,表明兩種抗原的制備是成功的。利用BCA法測得AMT-BSA和AMT-OVA的蛋白濃度分別為3.2 mg/mL和3.4 mg/mL。2.AMT單抗制備本實驗采取常規(guī)免疫方案免疫雌性Balb/c小鼠,用人工合成的AMT-BSA作為免疫原,以AMT-OVA為包被原。五次免疫后第7 d小鼠斷尾采血,測血清效價及抑制情況,檢測得血清效價達16000以上。利用細胞融合技術(shù)、ELISA篩選方法和有限稀釋法進行亞克隆獲得2株可持續(xù)分泌針對AMT抗體的雜交瘤細胞株,分別命名為1D11、2F9。采用小鼠體內(nèi)誘生法制備抗AMT單克隆抗體的腹水,其蛋白濃度分別為20.7mg/mL和25.3mg/mL;抗體效價分別為32000和128000。采用HiTrap Protein G HP純化柱純化腹水,純化后經(jīng)SDS-PAGE鑒定雜蛋白明顯減少。交叉實驗結(jié)果表明,該抗體與金剛烷、利巴韋林、嗎啉胍、阿莫西林、頭孢噻呋幾乎沒有交叉反應(yīng),與鹽酸金剛乙胺的交叉反應(yīng)率為15%,而對AMT的抑制率為100%。3.ELISA檢測方法的建立利用純化后的2F9單克隆抗體建立檢測AMT殘留的間接競爭ELISA方法。包被原最佳稀釋濃度為1:4000,抗體的最佳工作濃度為1:64000,AMT濃度在10～500ng/mL時,標準曲線線性良好,線性方程為y=0.3785x-0.2179(R2=0.9925),IC50為 77.83ng/mL,LOD為 5.98 ng/mL,LOQ 為 54.78 ng/mL。4.雞肉AMT添加回收實驗以雞肉進行添加AMT回收實驗,雞肉樣品經(jīng)過前處理后用于CiELISA測定。實驗結(jié)果表明,雞肉樣品處理液稀釋16倍以上時樣品的基質(zhì)干擾基本消失。在范圍10～1000ng/mL內(nèi),曲線標準方程式為 y=0.3377x-0.1658,相關(guān)系數(shù) R2=0.9901,IC50 為 92.67ng/mL,LOD為 6.74ng/mL,LOQ 為 132.82ng/mL。用 10、500 和 1000ng/mL 的 AMT 標準液做添加回收實驗,經(jīng)檢測雞肉樣品添加回收率在83.4%～102.3%之間。
[Abstract]:Amantadine (AMT) belongs to tricyclic amines. It is the earliest antiviral drug used in the treatment of influenza. It is mainly used for the prevention and treatment of avian influenza and transmissible gastroenteritis in livestock and poultry breeding. However, the side effects of mental disorders and the increasing drug resistance may cause more harm to consumers of animal products. Therefore, the use of amantadine and amantadine and other antiviral drugs in livestock and poultry farming has been prohibited by the announcement No. 560 of the Ministry of Agriculture of China, and the use of such drugs in livestock and poultry farming has been prohibited by FAD of the United States. Chromatography, liquid chromatography-tandem mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, hydrophilic interaction chromatography-tandem mass spectrometry and potentiometric titration, etc. However, traditional instrumental detection methods require not only expensive instruments and equipment, but also professional technicians to operate complex sample processing processes, which are caused by these disadvantages. ELISA is based on the specific binding of antigen and antibody. It has high selectivity and sensitivity. The method is low cost, simple, efficient, flexible and easy to carry. It is suitable for rapid screening of large quantities of samples in the field. Amantadine was synthesized by glutaraldehyde method. Balb/c mice were immunized after dialysis and identification. Monoclonal antibody technology, enzyme-linked immunosorbent assay (ELISA) and mouse ascites preparation were used to prepare monoclonal antibodies against amantadine with high specificity and titer. On this basis, an indirect competitive ELISA method for the detection of amantadine was established. 1. Glutaraldehyde is a bifunctional crosslinking agent. Its two aldehyde groups can form Schiff bases with primary amino groups on hapten and carrier proteins respectively. The two molecules are bridged by a five-carbon chain to synthesize a complete antigen. The complete antigen is scanned by ultraviolet spectrum. The complete antigen was successfully prepared by immunization, cell fusion and ELISA. The protein concentrations of AMT-BSA and AMT-OVA were 3.2 mg/ml and 3.4 mg/ml. AMT-BSA was synthesized as immunogen and AMT-OVA was used as coating agent. On the 7th day after five immunizations, the serum titer and inhibition were measured. The serum titer was more than 16 000. Two hybridoma cell lines secreting antibodies against AMT were obtained by cell fusion, ELISA screening and limited dilution. Ascites containing anti-AMT monoclonal antibodies were prepared by induction in vivo in mice with protein concentrations of 20.7 mg/mL and 25.3 mg/mL, and antibody titers of 32 000 and 128000, respectively. Ascites were purified by HiTrap Protein G-HP column, and the impurity proteins identified by SDS-PAGE were significantly reduced. There were almost no cross reactions with amantadine, ribavirin, morpholine guanidine, amoxicillin and ceftiofur. The cross reaction rate with amantadine hydrochloride was 15% and the inhibition rate against AMT was 100%. 3. Establishment of an indirect competitive ELISA method for detecting AMT residues using purified 2F9 monoclonal antibody. The linear equation was y=0.3785x-0.2179 (R2=0.9925), IC50 was 77.83ng/mL, LOD was 5.98 ng/mL, LOQ was 54.78 ng/mL. In the range of 10-1000ng/mL, the standard equation of the curve is y=0.3377x-0.1658, the correlation coefficient R2=0.9901, the IC50 is 92.67ng/mL, the LOD is 6.74ng/mL, the LOQ is 132.82ng/mL, and the AMT is 10,500 and 1000ng/mL. The recovery rate of chicken samples was between 83.4% and 102.3%.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S859.84

【參考文獻】

相關(guān)期刊論文 前10條

1 許靜;林翠鴻;劉亦偉;王長連;;UPLC-MS/MS法同時測定人血漿中普拉克索和金剛烷胺濃度[J];藥物分析雜志;2016年05期

2 張強;;ELISA技術(shù)在食品安全檢測中的應(yīng)用研究進展[J];長江大學(xué)學(xué)報(自科版);2015年33期

3 邱婧;童達;李曉燕;;氣相色譜法測定小兒氨酚烷胺顆粒中鹽酸金剛烷胺的含量[J];中國藥師;2015年06期

4 張立華;周劍;王敏;;中獸藥散劑中非法添加金剛烷胺的檢測方法研究及應(yīng)用[J];化學(xué)試劑;2015年05期

5 李豐清;;獸藥殘留的原因及防控措施[J];畜牧獸醫(yī)科技信息;2015年01期

6 徐逍;吳康;楊彩根;侍苗苗;宋學(xué)宏;;恩諾沙星多克隆抗體制備技術(shù)的優(yōu)化及其ELISA分析方法的建立[J];中國抗生素雜志;2014年12期

7 劉丹;孫卓;王洪媛;;金剛烷胺及其類似物抗帕金森病的研究進展[J];北方藥學(xué);2014年12期

8 胡巧茹;周長朋;張玉春;崔嘉;鞠玲燕;楊麗君;;高效液相色譜-串聯(lián)質(zhì)譜法測定辣椒及辣椒制品中金剛烷胺殘留[J];分析測試學(xué)報;2014年10期

9 吳銀良;趙健;葉宇飛;朱勇;孫亞米;;同位素稀釋液相色譜-串聯(lián)質(zhì)譜法測定雞蛋中金剛烷胺類藥物殘留量[J];分析測試學(xué)報;2014年08期

10 劉雪紅;董紫凌;韓鳳麗;鄭潔;馬慧楠;宋平;田曉玲;;超高效液相色譜-串聯(lián)質(zhì)譜法檢測清瘟敗毒散中非法添加金剛烷胺的研究[J];中國獸藥雜志;2014年08期

相關(guān)會議論文 前1條

1 劉華雷;王志亮;呂艷;鄭東霞;趙云玲;孫承英;張維;李金明;;不同亞型禽流感病毒對金剛烷胺的耐藥性分析[A];中國畜牧獸醫(yī)學(xué)會獸醫(yī)公共衛(wèi)生學(xué)分會第二次學(xué)術(shù)研討會論文集[C];2010年

相關(guān)博士學(xué)位論文 前1條

1 孫海新;雞肉中金剛烷胺殘留的生物識別材料研究與親和檢測方法建立[D];中國海洋大學(xué);2013年

相關(guān)碩士學(xué)位論文 前6條

1 吳松松;金剛烷胺免疫學(xué)快速定量檢測方法初步研究[D];南昌大學(xué);2016年

2 張立華;金剛烷胺溶液及雞肉粉基體標準物質(zhì)研制[D];中國農(nóng)業(yè)科學(xué)院;2015年

3 楊曉霞;金剛烷胺—油酸藥物囊泡的制備、理化性質(zhì)及其釋藥研究[D];山東大學(xué);2012年

4 張丹;淀粉糖化酶抗體的研制及其雙抗夾心ELISA法的初步建立與應(yīng)用[D];揚州大學(xué);2012年

5 楊令芝;M2離子通道中金剛烷胺的作用位點研究及BM2離子通道阻斷劑篩選[D];中國科學(xué)技術(shù)大學(xué);2009年

6 劉暢;動物源性食品中常見藥物殘留檢測方法的建立與應(yīng)用[D];第二軍醫(yī)大學(xué);2008年

，

本文編號：2210035