【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(qiáng)的耐藥性對(duì)畜產(chǎn)品消費(fèi)者可能造成的危害越來(lái)越引起關(guān)注。因此,我國(guó)農(nóng)業(yè)部560號(hào)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(guó)FAD也已明確禁止在畜禽養(yǎng)殖中使用此類藥物。檢測(cè)金剛烷胺的殘留可采用傳統(tǒng)的儀器檢測(cè)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(cè)方法不僅需要昂貴的儀器設(shè)備,復(fù)雜的樣品處理過(guò)程還需要專業(yè)的技術(shù)人員進(jìn)行操作,這些不利因素使這些檢測(cè)方法不適用于現(xiàn)場(chǎng)高通量的樣品檢測(cè)。ELISA檢測(cè)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(cè)方法成本低、簡(jiǎn)單、高效、靈活且便于攜帶,適合在現(xiàn)場(chǎng)大批量的樣品的快速篩選。本實(shí)驗(yàn)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(yàn)(ELISA)和小鼠腹水制備等技術(shù),制備出針對(duì)金剛烷胺的特異性強(qiáng)、效價(jià)高的單克隆抗體,并在此基礎(chǔ)上建立檢測(cè)金剛烷胺的間接競(jìng)爭(zhēng)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(gè)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(lái),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(xì)胞融合及ELISA測(cè)定,表明兩種抗原的制備是成功的。利用BCA法測(cè)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(shí)驗(yàn)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(cè)血清效價(jià)及抑制情況,檢測(cè)得血清效價(jià)達(dá)16000以上。利用細(xì)胞融合技術(shù)、ELISA篩選方法和有限稀釋法進(jìn)行亞克隆獲得2株可持續(xù)分泌針對(duì)AMT抗體的雜交瘤細(xì)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(jià)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(shí)驗(yàn)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(méi)有交叉反應(yīng),與鹽酸金剛乙胺的交叉反應(yīng)率為15%,而對(duì)AMT的抑制率為100%。3.ELISA檢測(cè)方法的建立利用純化后的2F9單克隆抗體建立檢測(cè)AMT殘留的間接競(jìng)爭(zhēng)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(shí),標(biāo)準(zhǔn)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(shí)驗(yàn)以雞肉進(jìn)行添加AMT回收實(shí)驗(yàn),雞肉樣品經(jīng)過(guò)前處理后用于CiELISA測(cè)定。實(shí)驗(yàn)結(jié)果表明,雞肉樣品處理液稀釋16倍以上時(shí)樣品的基質(zhì)干擾基本消失。在范圍10～1000ng/mL內(nèi),曲線標(biāo)準(zhǔn)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(biāo)準(zhǔn)液做添加回收實(shí)驗(yàn),經(jīng)檢測(cè)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(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S859.84

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