【摘要】：本論文優(yōu)化建立了恩諾沙星膠體金免疫層析分析方法,并有效用于動(dòng)物源性食品、環(huán)境樣品及動(dòng)物代謝物中恩諾沙星的快速檢測(cè)。 本研究利用混合酸酐法合成恩諾沙星包被原,將恩諾沙星抗血清經(jīng)protein A-sepharose4B進(jìn)行純化,得到抗恩諾沙星多克隆抗體。采用檸檬酸三鈉還原法制備粒徑為17nm膠體金,通過(guò)一系列優(yōu)化,最終確定膠體金連接金標(biāo)抗體最適pH為9,最佳標(biāo)記1mg/mL恩諾沙星多克隆抗體用量為20μL。根據(jù)優(yōu)化用量標(biāo)記恩諾沙星多克隆抗體,制得膠體金標(biāo)記抗體。以硝酸纖維素膜為固相載體,ENR-OVA包被原)和羊抗兔二抗分別作為檢測(cè)線和控制線包被在硝酸纖維素膜上,金標(biāo)記抗體噴涂在玻璃纖維墊上,制成快速檢測(cè)恩諾沙星免疫層析試紙條。 建立了膠體金免疫層析分析方法,檢出限為5μg/L,在濃度為1000μg/L時(shí),15種喹諾酮類藥物及8種常用獸藥對(duì)恩諾沙星免疫膠體金快速檢測(cè)均無(wú)明顯干擾,表明恩諾沙星膠體金免疫層析分析方法具有較高特異性。 選取牛奶、豬肉、豬肝等9種動(dòng)物源性產(chǎn)品、池塘水及豬尿作為檢測(cè)樣品,分別添加5,20,100μL的恩諾沙星標(biāo)準(zhǔn)品,經(jīng)過(guò)簡(jiǎn)單的處理即可上樣檢測(cè),牛奶、池塘水及豬尿樣品檢測(cè)限在5-25μg/L之間,其余動(dòng)物源性產(chǎn)品檢測(cè)限為50μg/kg,均低于最高殘留限量。所建立的恩諾沙星免疫層析分析方法與酶聯(lián)免疫分析方法的一致性較好,驗(yàn)證了方法的準(zhǔn)確性。 論文所開(kāi)發(fā)的膠體金試紙條無(wú)需檢測(cè)儀器,操作簡(jiǎn)單,快速,準(zhǔn)確。實(shí)驗(yàn)結(jié)果穩(wěn)定性高,可作為動(dòng)物性食品中恩諾沙星殘留現(xiàn)場(chǎng)大量篩選,及監(jiān)測(cè)動(dòng)物生長(zhǎng)環(huán)境中恩諾殺星濫用的有效手段。
[Abstract]:In this paper, a gold immunochromatography method of enrofloxacin colloid was established and applied to the rapid detection of enrofloxacin in animal-derived food, environmental samples and animal metabolites. Enrofloxacin coated proto was synthesized by mixed anhydride method in this study. Enrofloxacin antiserum was purified by protein A-sepharose4B to obtain polyclonal antibody against enrofloxacin. The particle size of 17nm colloidal gold was prepared by tricarbonate reduction method. Through a series of optimization, the optimal pH and 1mg/mL enrofloxacin polyclonal antibody dosage were determined to be 9 and 20 渭 L for colloidal gold conjugated gold antibody and enrofloxacin polyclonal antibody respectively. Colloidal gold labeled antibody was prepared by labeling enrofloxacin polyclonal antibody according to the optimal dosage. Using nitrocellulose membrane as solid phase carrier and sheep anti-rabbit second antibody as detection line and control line respectively, the nitrocellulose membrane was coated on the nitrocellulose membrane, and the gold labeled antibody was sprayed on the glass fiber pad, respectively, using the ENR-OVA coating as the carrier and the sheep anti-rabbit second antibody as the test line and control line respectively. The rapid detection of enrofloxacin immunochromatographic test strip was made. A method of colloidal gold immunochromatography was established. The detection limit was 5 渭 g / L. When the concentration was 1 000 渭 g / L, 15 quinolones and 8 common veterinary drugs did not interfere with the rapid detection of enrofloxacin immunogold. The results showed that the gold immunochromatographic assay of enrofloxacin colloid was highly specific. Nine kinds of animal-derived products such as milk, pork, pig liver, pond water and pig urine were selected as detection samples, and enrofloxacin standard samples of 5,20100渭 L were added respectively. After simple treatment, samples can be detected, milk, etc. The detection limit of pond water and pig urine was 5-25 渭 g / L, and the detection limit of other animal-derived products was 50 渭 g / kg, lower than the maximum residue limit. The established method of enrofloxacin immunochromatographic assay was in good agreement with that of enzyme-linked immunosorbent assay, and the accuracy of the method was verified. The colloidal gold test strip developed in this paper does not require a detection instrument, and is simple, rapid and accurate. The results were stable and could be used as an effective method to screen the residues of enrofloxacin in animal food and monitor the abuse of enrofloxacin in animal growing environment.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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本文編號(hào)：2464818