莱克多巴胺荧光微球免疫层析快速检测方法的建立

发布时间：2018-05-16 04:02

本文选题：莱克多巴胺 + 免疫层析　；参考：《暨南大学》2012年硕士论文

【摘要】：目的：依据免疫竞争层析方法的原理，建立莱克多巴胺（RAC）荧光微球免疫层析快速检测方法。研制莱克多巴胺荧光微球免疫层析试纸条，用于猪尿中莱克多巴胺残留的检测。方法：采用饱和硫酸铵沉淀法纯化抗RAC单抗小鼠腹水，并用BCA试剂盒测定其抗体蛋白浓度。以EDC作为偶联剂，将本实验室制备的抗RAC单抗偶联到商用聚苯乙烯荧光微球表面，合成出荧光微球抗体复合物。将标记好的复合物喷涂于结合垫上；采用活性酯法合成RAC-BSA检测抗原并将其包被在NC膜表面作为检测线(T线)；羊抗鼠IgG包被在硝酸纤维素（NC）膜表面作为质控线(C线)；筛选并优化复溶液及样品垫预处理液中的各项成分及浓度。将结合垫、吸水纸和处理过的样品垫最后组装成试纸条。试纸条检测过程中RAC-BSA与待测样品中RAC竞争结合有限的荧光微球标记RAC单抗，如果样品中的RAC含量多则其结合荧光微球标记的抗体多，则结合到T线的荧光微球抗体复合物越少，于450nm激发光下观察，则T线的荧光强度越低，因此可根据荧光强度的强弱检测判断样品中RAC的多寡。结果：合成出检测抗原RAC-BSA。合成得到了荧光微球抗体复合物，该复合物与检测抗原有很好的结合，且与BSA不存在非特异性结合。建立了RAC荧光快速检测方法，制备出了RAC荧光微球免疫层析试纸条，该试纸条检测猪尿中RAC的残留，，灵敏度最低值为2ngmL~(-1)，与克伦特罗（CL），沙丁胺醇（SAL）等类似物无交叉反应。结论：本研究建立的莱克多巴胺荧光微球免疫层析快速检测技术操作便捷，稳定可靠，灵敏度高，可作为猪尿中莱克多巴胺残留现场检测和监控的有效手段。
[Abstract]:Aim: to establish a rapid immunochromatographic method for detection of ractopamine rac fluorescent microspheres according to the principle of immunocompetitive chromatography. The ractopamine fluorescent microspheres immunochromatographic strip was developed for the detection of ractopamine residues in pig urine. Methods: the mouse ascites of anti RAC monoclonal antibody were purified by saturated ammonium sulfate precipitation method and the concentration of antibody protein was determined by BCA kit. Using EDC as coupling agent, the anti RAC monoclonal antibody prepared in our laboratory was coupled to the surface of commercial polystyrene fluorescent microspheres to synthesize fluorescent microspheres antibody complexes. Spraying the labeled composite onto the binding pad; RAC-BSA detection antigen was synthesized by active ester method and coated on the surface of NC membrane as T line; goat anti rat IgG coating on nitrocellulose membrane surface was used as quality control line C line; complex solution and sample pad pretreatment were selected and optimized. The composition and concentration of the liquid. Combine mats, absorbent paper and treated mats to form test strips. In the process of test strip detection, RAC-BSA competes with RAC in the test sample to bind to the limited fluorescent microsphere labeled RAC McAb. If the sample contains more RAC, it binds to the fluorescent microsphere antibody more, then the less fluorescent microsphere antibody complex binds to the T line. Observed under 450nm excitation light, the fluorescence intensity of T line is lower. Therefore, the amount of RAC in the sample can be judged by the detection of fluorescence intensity. Results: the antigen RAC-BSA was synthesized. The fluorescent microsphere antibody complex was synthesized, which binds well to the detection antigen and has no nonspecific binding to BSA. A rapid RAC fluorescence detection method was established, and a RAC fluorescent microsphere immunochromatographic strip was prepared. The test strip was used to detect RAC residues in porcine urine. The lowest sensitivity was 2ngmLnLX, and there was no cross reaction with clenbuterol and clenbuterol (clenbuterol) and the other analogs, such as clenbuterol or clenbuterol. Conclusion: the rapid detection technique of ractopamine fluorescence microsphere immunochromatography is easy to operate, stable, reliable and sensitive. It can be used as an effective method for detection and monitoring of ractopamine residues in pig urine.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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