本文選題：膠體金免疫層析試紙條 + 玉米赤霉烯酮��； 參考：《南昌大學》2015年碩士論文

【摘要】：玉米赤霉烯酮(zearalenone,ZEN)是由鐮刀菌所產(chǎn)生的具有類雌激素作用的次級代謝產(chǎn)物。ZEN對食品和飼料的污染范圍較為廣泛,動物和人體攝入后,對機體具有一定的毒害作用,對畜牧養(yǎng)殖業(yè)和人體健康會造成很大的損害。因此,加強對食品及飼料中ZEN的監(jiān)控檢測顯得尤為重要。由于飼料樣本存在一定的基質干擾,對定量檢測的結果會造成一定的影響,本文采用了聚乙烯亞胺-戊二醛介導體系制備了ZEN免疫磁性微球,建立了高效富集捕獲飼料樣本中ZEN的方法,以降低樣本的基質效應對檢測結果的影響。再以膠體金作為標記材料,研制出了膠體金免疫層析試紙條,結合HG-8膠體金試紙條讀取儀,實現(xiàn)了對飼料中ZEN殘留的快速定量檢測。通過DCC/NHS活潑酯法制備了ZEN-BSA人工全抗原,并通過TLC監(jiān)控和紫外全波段掃描驗證ZEN-BSA成功合成,ZEN-BSA的濃度為2.55 mg/m L;用辛酸-硫酸銨法純化了抗ZEN單抗,對抗體的性能進行了評價,測得效價為1:160000,濃度為5.36 mg/mL,通過SDS-PAGE電泳對純化后的單抗進行了純度分析,結果表明該單抗具有較高的純度。為了保證捕獲效率,本文以聚乙烯亞胺-戊二醛介導的方法制備了免疫磁性微球,對抗ZEN單克隆抗體的偶聯(lián)量進行了優(yōu)化,最佳偶聯(lián)條件為:1 mg磁球表面偶聯(lián)抗體100μg;對免疫磁富集捕獲緩沖液的pH、甲醇濃度、捕獲時間、微球加入量、洗脫液種類等條件進行了優(yōu)化,最佳條件如下:將200μg PEI免疫磁球加入1 mL 20%甲醇PBS捕獲緩沖液(pH7.0)中,在劇烈振蕩條件下提取10 min,磁分離后用100μL 3%氨化甲醇洗脫磁球上的ZEN,洗脫液稀釋復溶后用ELISA試劑盒檢測。通過樣本加標回收試驗的結果表明,當ZEN加標量為50-250μg/kg時回收率逐漸上升,且在250μg/kg時,回收率達到最高,為93.28%。本文以膠體金作為標記物,優(yōu)化了膠體金的粒徑、抗體標記的pH值、抗體的標記量、T線全抗原的包被濃度、C線二抗的包被濃度和金標抗體的噴量等幾個參數(shù),確定了定量檢測ZEN膠體金免疫層析試紙條的最佳生產(chǎn)工藝條件:膠體金粒徑為40 nm,標記pH為6.0,抗體標記量為4μg/mL,結合墊上的金標抗體噴量為2μL/cm,NC膜上T線ZEN-BSA的包被濃度為0.8 mg/mL,C線上驢抗鼠二抗的包被濃度為0.4 mg/mL。由此,成功地研制出ZEN膠體金免疫層析定量檢測試紙條,定量檢測時間為15 min,該試紙條具有良好的特異性和穩(wěn)定性,最低靈敏度為2.559μg/L,IC50=9.09μg/L。加標回收率在67.70%—104.48%之間。此外,采用ELISA試劑盒評估了本法的準確性,兩種方法的相關性較好。
[Abstract]:Zen, a secondary metabolite produced by Fusarium oxysporum, has a wide range of contamination of food and feed. After ingested by animals and human beings, Zen has a certain toxic effect on the body. It will do great harm to animal husbandry and human health. Therefore, it is particularly important to strengthen the monitoring and detection of ZEN in food and feed. ZEN immunomagnetic microspheres were prepared by polyethyleneimide-glutaraldehyde (Glutaraldehyde) mediated system because of some matrix interference in feed samples. In order to reduce the effect of matrix effect on the detection results, a method of high efficiency enrichment and capture of ZEN in feed samples was established. Using colloidal gold as labeling material, a colloidal gold immunochromatographic strip was developed. Combined with HG-8 colloidal gold strip reader, the rapid quantitative determination of ZEN residues in feed was realized. The artificial antigens of ZEN-BSA were prepared by DCC/NHS active ester method, and the concentration of ZEN-BSA was 2.55 mg/m L by TLC monitoring and UV scanning. The anti-ZEN monoclonal antibody was purified by octanoic acid-ammonium sulfate method, and the performance of the antibody was evaluated. The titer was 1: 1600 and the concentration was 5.36 mg / mL. The purity of the purified McAb was analyzed by SDS-PAGE electrophoresis. The results showed that the McAb had high purity. In order to ensure the capture efficiency, the immunomagnetic microspheres were prepared by polyethyleneimide-glutaraldehyde mediated method, and the coupling amount of monoclonal antibody against ZEN was optimized. The optimal coupling condition was 100 渭 g for the surface coupling antibody of magnetic sphere at 1 mg, the pH of the magnetic enrichment capture buffer, the concentration of methanol, the capture time, the amount of microspheres and the type of eluent were optimized. The optimum conditions were as follows: 200 渭 g PEI immunomagnetic sphere was added to 1 mL 20% methanol PBS capture buffer (pH 7.0), extracted under the condition of intense oscillation for 10 min, then separated by magnetic separation and eluted by 100 渭 L 3% amination methanol on the magnetic sphere. The eluent was diluted and redissolved and detected by ELISA kit. The results of the standard recovery test showed that the recovery rate increased gradually when the scalar concentration of ZEN was 50-250 渭 g/kg, and the recovery rate reached the highest level at #number0# 渭 g/kg, 93.28 渭 mol 路mol ~ (-1) 路L ~ (-1) 路min ~ (-1) 路min ~ (-1). In this paper, the colloidal gold was used as a marker to optimize the particle size of colloidal gold, the pH value of antibody labeling, the concentration of T line whole antigen coated with antibody and the coating concentration of second antibody of C line and the amount of spray of gold labeled antibody. The optimum production conditions for quantitative detection of ZEN colloidal gold immunochromatographic strip were determined as follows: colloidal gold particle size was 40 nm, labeling pH was 6.0, antibody labeling amount was 4 渭 g / mL, and the amount of gold-labeled antibody sprayed on the pad was 2 渭 L / cm ~ (-1) of T line ZEN-BSA coated on NC membrane. The coating concentration of the donkeys anti mouse second antibody on the line of 0.8 mg / mL C was 0.4 mg / mL. Therefore, the ZEN colloidal gold immunochromatographic quantitative test strip was successfully developed. The quantitative detection time was 15 min. The test strip had good specificity and stability, and the lowest sensitivity was 2.559 渭 g / L IC50 / 9 渭 g / L, and the minimum sensitivity was 2.559 渭 g / L ~ (50) and 9.09 渭 g 路L ~ (-1) 路L ~ (-1). The recoveries were between 67.70% and 104.48%. In addition, the ELISA kit was used to evaluate the accuracy of the two methods.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S816;O652

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