四环素酶联免疫检测方法的研究

发布时间：2019-04-02 05:10

【摘要】：本论文对检测动物源性食品中四环素残留的间接竞争酶联免疫检测方法进行了系统的研究。采用甲醛法将四环素与钥孔血蓝蛋白偶联制备人工免疫原。通过免疫新西兰大白兔得到抗血清,使用Protein A Sepharose-4B亲和层析法纯化血清得到高效价和高特异性的抗四环素多克隆抗体。采用甲醛法将四环素与卵血清白蛋白连接制备包被原,建立了间接竞争酶联免疫检测方法,对影响实验的各因素(包被量,抗体稀释度,抗体稀释液等)进行了优化与研究,最终确定包被量为0.05¨μgg/孔,抗体稀释度为1：10000,抗体稀释液为0.1%BSA/PBS,封闭液为0.5%乳粉/PBS溶液时所获得的标准曲线灵敏度最高,稳定性最好。本实验所建立方法的灵敏度(IC50)为0.33±0.09μg/L,最低检出限(IC15)为0.01±0.0041μg/L。本实验所制备的抗体具有良好的特异性,与两种结构类似物土霉素、金霉素交叉反应率分别为0.39%和4.42%,与其他常用兽药青霉素、链霉素、氯霉素均无交叉反应。选择牛奶、蜂蜜、猪肉、牛肉、鸡肉、鱿鱼、大眼鲷鱼、比目鱼、虾作为样品进行添加回收实验,牛奶添加回收率为85.88%-109.17%,蜂蜜添加回收率为94.52%-107.59%,猪肉添加回收率为84.19%-98.96%,牛肉添加回收率为75.91%-105.58%,鸡肉添加回收率为84.40%-109.64%,鱿鱼添加回收率为73.08%-115.44%,大眼鲷鱼添加回收率为67.45%-104.76%,比目鱼添加回收率为74.55%-112.46%,虾添加回收率为82.84%-111.85%。本实验所建立的方法在牛奶、鱿鱼中检出限为5μg/kg,蜂蜜、猪肉、牛肉、鸡肉、大眼鲷鱼、比目鱼、虾检出限为10μg/kg,板内变异和板间变异均小于20%。使用国外试剂盒验证本实验建立的四环素间接竞争酶联免疫法,两种方法的检测结果一致性很高,其线性回归方程为：y=0.8542x+2.2848,线性相关系数R2=0.9954。说明本研究建立的方法具有很好的准确性和精密度。本研究所建立的四环素间接竞争酶联免疫检测方法灵敏度高、特异性好、准确性高、样品前处理简单,适合于动物源性食品中四环素残留的快速检测。
[Abstract]:In this paper, indirect competitive enzyme-linked immunosorbent assay (Elisa) for the detection of tetracycline residues in animal-derived foods was systematically studied. Artificial immunogen was prepared by coupling tetracycline with keyhole hemocyanin by formaldehyde method. Anti-tetracycline polyclonal antibodies were obtained by immunizing New Zealand white rabbits and purified by Protein-A-Sepharose-4B affinity chromatography. An indirect competitive enzyme-linked immunosorbent assay (Elisa) was established by linking tetracycline with ovalbumin by formaldehyde method. The factors influencing the experiment, such as coating quantity, antibody dilution, antibody diluent, etc., were optimized and studied. The best stability and sensitivity of the standard curve were obtained when the coating volume was 0.05 渭 gg/, the dilution of antibody was 1 脳 10 000, the diluent of antibody was 0.1% BSA / PBS, and the blocking solution was 0.5% milk powder / PBS. the results showed that the best stability and sensitivity of the standard curve were obtained when the coating volume was 0.05 渭 mol / min, the dilution of antibody was 1? The sensitivity (IC50) and the minimum detection limit (IC15) of the method were 0.33 卤0.09 渭 g / L and 0.01 卤0.0041 渭 g / L, respectively. The cross-reaction rates with oxytetracycline and chloramphenicol were 0.39% and 4.42%, respectively. There was no cross-reaction with other commonly used veterinary drugs such as penicillin, streptomycin and chloramphenicol. Choose milk, honey, pork, beef, chicken, squid, big-eyed snapper, halibut, shrimp as samples to add and recycle experiment, milk added recovery rate is 85.88% and 109.17%. The recoveries of honey, pork, beef and chicken were 94.52% and 107.59%, 84.19%, 98.96%, 75.91% and 105.58%, 84.40% and 109.64%, respectively. The recovery of squid and bream was 73.08% / 115.44%, 67.45% / 104.76%, 74.55% / 112.46% and 82.84% / 111.85%, respectively. The detection limit of this method in milk and squid was 5 渭 g / kg, honey, pork, beef, chicken, snapper, halibut and shrimp. The detection limit was 10 渭 g / kg, intraplate variation and interplate variation less than 20%. The test results of tetracycline indirect competitive enzyme-linked immunosorbent assay (Elisa) were verified with foreign kits. The results of the two methods were consistent with each other. The linear regression equation was y = 0.8542 x 2.2848, and the linear correlation coefficient (R _ (2) was R _ (2) = 0.9954. It shows that the method established in this study has good accuracy and precision. The indirect competitive enzyme-linked immunosorbent assay for tetracycline detection developed in this study has high sensitivity, specificity, high accuracy and simple pretreatment. It is suitable for the rapid detection of tetracycline residues in animal-derived foods.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R155.5

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