链霉素酶联免疫分析方法的研究

发布时间：2018-05-10 16:26

本文选题：链霉素 + 残留　；参考：《天津科技大学》2013年硕士论文

【摘要】：本文针对动物源性食品和饲料中链霉素残留潜在的安全问题,研究建立了间接竞争酶联免疫检测方法(icELISA)。本研究采用环氧树脂法将链霉素(STR)与牛血清蛋白(BSA)偶联制得免疫原,免疫新西兰白兔,得到链霉素多克隆抗体。通过链霉素(STR)与O-羧甲基羟胺(CMO)连接,引入羧基活性基团成功得到半抗原STR-CMO,并利用碳二亚胺法将半抗原与卵清蛋白(OVA)偶联制得包被抗原。通过优化包被原包被量,抗体稀释倍数,离子强度,pH值等参数,建立了间接竞争酶联免疫检测方法。最终选择抗体稀释倍数为1：1000；包被抗原的包被量为0.5 μg/well;封闭液为0.5%脱脂乳粉/PBS；样品稀释液为0.01 mol/L PBS,pH 7.4;抗体稀释液为0.1%BSA/PBS。所建立的icELISA方法灵敏度(IC50)为2.00±0.24 ng/mL,检测限(IC15)为0.24±0.08 ng/mL。所制备的链霉素抗体对双氢链霉素的交叉反应为95.50%,对其他六种结构类似的氨基糖苷类抗生素没有交叉反应,证明此抗体具有很高的特异性。选取了鸡肉、鸡肝、猪肉、猪肝、猪肾、鱼肉、虾肉、牛奶、鸡饲料和猪饲料作为样品进行添加回收实验。样品在15ng/g、30ng/g、120ng/g、300ng/g、600ng/g五个添加水平上的回收率在71.32-106.94%之间,板内及板间变异系数均小于18%。回收率的方差分析表明,除肝脏样品外,对于不同的链霉素残留量以及不同类型的检测样品,方法的回收率之间没有显著性差异。通过与商品化试剂盒进行比较,二者的检测结果具有较好的相关性,线性相关系数R2为0.9854,且本实验所建立的方法针对动物组织样品的检出限优于商品化试剂盒。说明本方法具有很好的准确度和广泛的应用性。
[Abstract]:An indirect competitive enzyme-linked immunosorbent assay (Elisa) for the detection of streptomycin residues in animal derived food and feed was developed in this paper. In this study, the immunogen was prepared by coupling streptomycin (STR) with bovine serum protein (BSA) by epoxy resin method. New Zealand white rabbits were immunized with streptomycin polyclonal antibody. The hapten STR-CMOs were successfully obtained by linking streptomycin (STR) with O-carboxymethyl hydroxylamine (CMO), and the coated antigen was prepared by coupling hapten with ovalbumin (OVA) by carbodiimide method. An indirect competitive enzyme-linked immunosorbent assay (Elisa) was established by optimizing the parameters such as the original coating volume, the dilution multiple of antibody and the pH value of ionic strength. Finally, the dilution ratio of antibody was 1: 1000, the coating amount of antigen was 0.5 渭 g / wellwell, the blocking solution was 0.5% defatted milk powder / PBS, the sample diluent was 0.01 mol/L PBSs pH 7.4, and the antibody dilution solution was 0.1 BSA-PBSs. The sensitivity of the icELISA method is 2.00 卤0.24 ng / mL, and the detection limit is 0.24 卤0.08 ng / mL. The cross reaction of streptomycin antibody against dihydrostreptomycin was 95.50, and there was no cross reaction with other six similar aminoglycoside antibiotics, which proved that the antibody had a high specificity. Chicken, chicken liver, pork, pig liver, pig kidney, fish meat, shrimp meat, milk, chicken feed and pig feed were selected as samples. The recoveries of the samples were 71.32-106.94% at the five additive levels of 15ng / g ~ 30ng / g ~ 120ng / g ~ 300ng / g / g ~ 600ng / g, and the coefficients of variation were less than 18% both intra- and inter-plate. The variance analysis of recovery showed that there was no significant difference between the recovery rates for different streptomycin residues and different types of samples except liver samples. The linear correlation coefficient R2 was 0.9854, and the detection limit for animal tissue samples was better than that of commercial kit. It shows that this method has good accuracy and wide application.
【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

【参考文献】