本文選題：免疫層析方法 + 定量檢測　； 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：近些年來,食品安全問題(獸藥殘留、農(nóng)藥殘留、違禁化合物添加和致病菌污染等)引起了人們的廣泛關(guān)注。與此同時,各項食品安全檢測技術(shù)也受到了社會的普遍關(guān)注。與傳統(tǒng)的儀器檢測方法相比,基于抗原-抗體特異性結(jié)合的免疫層析技術(shù)具有靈敏、快速、高通量、低成本、適于現(xiàn)場篩查等特點。早期的免疫層析檢測通過裸眼觀察僅能得到定性或半定量的檢測結(jié)果。隨著光學(xué)檢測儀器的發(fā)展,免疫層析技術(shù)結(jié)合便攜式光學(xué)讀取儀可實現(xiàn)高靈敏定量檢測目標(biāo)物。一些具有獨特光吸收或熒光光譜特性的新型納米粒子作為標(biāo)記物應(yīng)用在免疫層析技術(shù)中同樣可以提高檢測靈敏度或降低檢測限。目前免疫層析技術(shù)已成為食品安全中的重要檢測手段。本文第二章建立了一種定量檢測氟苯尼考的膠體金免疫層析方法。對膠體金標(biāo)記抗體時溶液pH和抗體濃度、金標(biāo)抗體用量、檢測線上抗原濃度以及檢測時間進(jìn)行了優(yōu)化。通過膠體金試紙條讀取儀記錄試紙條上檢測線(Test line,T line)和質(zhì)控線(Control line,C line)的信號強(qiáng)度,以氟苯尼考標(biāo)準(zhǔn)品的濃度為橫坐標(biāo),以BX/B0為縱坐標(biāo)為縱坐標(biāo)建立標(biāo)準(zhǔn)曲線,其中BX表示陽性樣本的檢測線信號強(qiáng)度(Absorbance of T line,AT)與質(zhì)控線信號強(qiáng)度(Absorbance of C line,AC)的比值(AT/AC),B0表示陰性樣本的AT/AC。結(jié)果表明:氟苯尼考膠體金免疫層析快速定量檢測法的線性范圍為0.1~1.5 ng/mL,檢測限為0.08ng/mL,檢測時間為15 min。本檢測方法具有簡便、快速和可定量等特點,適合于大批量樣品的現(xiàn)場篩查。本文第三章研究不同標(biāo)記物對免疫層析方法靈敏度的影響,系統(tǒng)地探討了基于免疫競爭模式的時間分辨熒光微球免疫層析方法(Time-resolved fluorescent nanobeads based-lateral flow assay,TRFN-LFA)、熒光微球免疫層析方法(Fluorescent microspheres based-lateral flow assay,FM-LFA)、量子點免疫層析方法(Quantum dots based-lateral flow assay,QD-LFA)和膠體金免疫層析方法(Colloidal gold based-lateral flow assay,CG-LFA)在定量檢測豬尿中的萊克多巴胺(Ractopamine,RAC)中的應(yīng)用,確定了四種免疫層析方法的最優(yōu)工藝參數(shù)。TRFN-LFA、FM-LFA、QD-LFA和CG-LFA四種免疫層析方法在豬尿樣本中的檢測限分別為7.2 pg/mL、14.7 pg/mL、23.6 pg/mL和40.1 pg/mL,其中TRFN-LFA的靈敏度是最高的。在定量檢測豬尿樣本中RAC時,TRFN-LFA具有較寬的線性范圍為5 pg/mL~2500 pg/mL,同時具有良好的相關(guān)系數(shù)(R2=0.9803)。FM-LFA、QD-LFA和CG-LFA三種免疫層析方法的線性范圍相對較窄,分別為10 pg/mL~500 pg/mL、25 pg/mL~2500 pg/mL和25 pg/mL~2500pg/mL。每一個TRFN-LFA試紙條只需要0.005μg的抗萊克多巴胺多克隆抗體(Anti-ractopamine polycolonal antibody,anti-RAC pAb),然而每一個FM-LFA、QD-LFA和CG-LFA試紙條需要的anti-RAC pAb分別為0.02μg、0.054μg和0.15μg。除此之外,與FM-LFA、QD-LFA和CG-LFA這三種免疫層析方法相比TRFN-LFA需要最少的RAC-BSA抗原,并且所需檢測時間最短。用TRFN-LFA分析豬尿樣本中的萊克多巴胺其檢測結(jié)果與液相色譜串聯(lián)質(zhì)譜法和商業(yè)用的酶聯(lián)免疫試劑盒測定的結(jié)果均一致。結(jié)果表明TRFN-LFA在檢測豬尿中的RAC時具有獨特的優(yōu)勢,TRFN-LFA在食品安全小分子物質(zhì)檢測方面有潛在的應(yīng)用價值。
[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.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TS207.5;O658.1

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