免疫层析定量检测氟苯尼考和莱克多巴胺的研究

发布时间：2018-05-08 14:32

本文选题：免疫层析方法 + 定量检测　；参考：《南昌大学》2017年硕士论文

【摘要】：近些年来,食品安全问题(兽药残留、农药残留、违禁化合物添加和致病菌污染等)引起了人们的广泛关注。与此同时,各项食品安全检测技术也受到了社会的普遍关注。与传统的仪器检测方法相比,基于抗原-抗体特异性结合的免疫层析技术具有灵敏、快速、高通量、低成本、适于现场筛查等特点。早期的免疫层析检测通过裸眼观察仅能得到定性或半定量的检测结果。随着光学检测仪器的发展,免疫层析技术结合便携式光学读取仪可实现高灵敏定量检测目标物。一些具有独特光吸收或荧光光谱特性的新型纳米粒子作为标记物应用在免疫层析技术中同样可以提高检测灵敏度或降低检测限。目前免疫层析技术已成为食品安全中的重要检测手段。本文第二章建立了一种定量检测氟苯尼考的胶体金免疫层析方法。对胶体金标记抗体时溶液pH和抗体浓度、金标抗体用量、检测线上抗原浓度以及检测时间进行了优化。通过胶体金试纸条读取仪记录试纸条上检测线(Test line,T line)和质控线(Control line,C line)的信号强度,以氟苯尼考标准品的浓度为横坐标,以BX/B0为纵坐标为纵坐标建立标准曲线,其中BX表示阳性样本的检测线信号强度(Absorbance of T line,AT)与质控线信号强度(Absorbance of C line,AC)的比值(AT/AC),B0表示阴性样本的AT/AC。结果表明:氟苯尼考胶体金免疫层析快速定量检测法的线性范围为0.1~1.5 ng/mL,检测限为0.08ng/mL,检测时间为15 min。本检测方法具有简便、快速和可定量等特点,适合于大批量样品的现场筛查。本文第三章研究不同标记物对免疫层析方法灵敏度的影响,系统地探讨了基于免疫竞争模式的时间分辨荧光微球免疫层析方法(Time-resolved fluorescent nanobeads based-lateral flow assay,TRFN-LFA)、荧光微球免疫层析方法(Fluorescent microspheres based-lateral flow assay,FM-LFA)、量子点免疫层析方法(Quantum dots based-lateral flow assay,QD-LFA)和胶体金免疫层析方法(Colloidal gold based-lateral flow assay,CG-LFA)在定量检测猪尿中的莱克多巴胺(Ractopamine,RAC)中的应用,确定了四种免疫层析方法的最优工艺参数。TRFN-LFA、FM-LFA、QD-LFA和CG-LFA四种免疫层析方法在猪尿样本中的检测限分别为7.2 pg/mL、14.7 pg/mL、23.6 pg/mL和40.1 pg/mL,其中TRFN-LFA的灵敏度是最高的。在定量检测猪尿样本中RAC时,TRFN-LFA具有较宽的线性范围为5 pg/mL~2500 pg/mL,同时具有良好的相关系数(R2=0.9803)。FM-LFA、QD-LFA和CG-LFA三种免疫层析方法的线性范围相对较窄,分别为10 pg/mL~500 pg/mL、25 pg/mL~2500 pg/mL和25 pg/mL~2500pg/mL。每一个TRFN-LFA试纸条只需要0.005μg的抗莱克多巴胺多克隆抗体(Anti-ractopamine polycolonal antibody,anti-RAC pAb),然而每一个FM-LFA、QD-LFA和CG-LFA试纸条需要的anti-RAC pAb分别为0.02μg、0.054μg和0.15μg。除此之外,与FM-LFA、QD-LFA和CG-LFA这三种免疫层析方法相比TRFN-LFA需要最少的RAC-BSA抗原,并且所需检测时间最短。用TRFN-LFA分析猪尿样本中的莱克多巴胺其检测结果与液相色谱串联质谱法和商业用的酶联免疫试剂盒测定的结果均一致。结果表明TRFN-LFA在检测猪尿中的RAC时具有独特的优势,TRFN-LFA在食品安全小分子物质检测方面有潜在的应用价值。
[Abstract]:In recent years, food safety problems (veterinary drug residues, pesticide residues, prohibited compounds added and pathogenic bacteria pollution) have aroused widespread concern. At the same time, various food safety detection techniques have also received widespread attention. Compared with traditional instrument detection methods, the immunochromatography technique based on antigen antibody specific combination It has the characteristics of sensitive, rapid, high throughput, low cost and suitable for on-site screening. Early immunochromatography detection can only obtain qualitative or semi quantitative detection results through naked eye observation. With the development of optical detection instruments, immunochromatography technique combined with portable optical reader can achieve high sensitivity and quantitative detection targets. New nanoparticles with special light absorption or fluorescence spectra can also be used as markers in immunochromatographic techniques to improve detection sensitivity or reduce detection limits. At present, immunochromatography has become an important detection method in food safety. In the second chapter, a colloidal gold immunochromatography for the determination of florfenicol was established in this paper. Methods. The concentration of pH and antibody, the amount of gold antibody, the concentration of the antigen and the detection time were optimized. The signal intensity of the detection line (Test line, T line) and the quality control line (Control line, C line) on the test paper was recorded by colloidal gold test paper reader. The concentration of the standard of florfenicol was used as the concentration of the test paper. A standard curve is established by BX/B0 as a longitudinal coordinate, in which BX indicates the ratio of the signal intensity of the detection line (Absorbance of T line, AT) to the signal intensity of the quality control line (Absorbance of C line, AC). The negative sample results show that the fast quantitative detection method of the florfenicol colloidal gold immunochromatography The linear range is 0.1~1.5 ng/mL, the detection limit is 0.08ng/mL, the detection time is 15 min., and the detection method is simple, fast and quantitative. It is suitable for the field screening of large quantities of samples. The third chapter studies the influence of different markers on the sensitivity of the immunochromatography method, and systematically discusses the time based on the immune competition mode. Time-resolved fluorescent nanobeads based-lateral flow assay (TRFN-LFA), fluorescent microsphere immunochromatography (Fluorescent microspheres based-lateral flow assay), quantum dot immunization chromatography and colloidal gold immunochromatography The method (Colloidal gold based-lateral flow assay, CG-LFA) was used in quantitative detection of Ractopamine (RAC) in porcine urine. The optimal technological parameters of four immunochromatographic methods,.TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA, were determined to be 7.2, 14.7, 23.6 and 14.7 respectively. G/mL and 40.1 pg/mL, of which the sensitivity of TRFN-LFA is the highest. In the quantitative detection of RAC, TRFN-LFA has a wider linear range of 5 pg/mL~2500 pg/mL, with a good correlation coefficient (R2=0.9803).FM-LFA, QD-LFA and CG-LFA three immunochromatography methods are relatively narrow, respectively 10 pg/mL~500, 2 Each TRFN-LFA test paper for 5 pg/mL~2500 pg/mL and 25 pg/mL~2500pg/mL. requires only 0.005 mu g for the anti lax dopamine polyclonal antibody (Anti-ractopamine polycolonal antibody, anti-RAC pAb). The three immunochromatographic methods, D-LFA and CG-LFA, need the least RAC-BSA antigen and have the shortest detection time. The results of the detection of ractopamine in the porcine urine samples by TRFN-LFA are in accordance with the results measured by the liquid chromatography tandem mass spectrometry and the commercial ELISA kit. The results showed that TRFN-LFA was detected. It has unique advantages to detect RAC in pig urine, and TRFN-LFA has potential application in detection of small molecules in food safety.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS207.5;O658.1

【参考文献】