【作者】 侯凌燕；

【导师】 章竹君；

【作者基本信息】 陕西师范大学 ， 分析化学， 2007， 硕士

【摘要】 本论文由综述、研究报告两部分组成。综述分别介绍了化学发光免疫分析的主要类型、新方法和化学发光免疫成像分析的历史发展和应用前景。化学发光免疫分析是将发光物质或酶标记在抗原或抗体上，免疫反应后，通过化学发光反应来测定发光标记物或酶标记物的化学发光信号，从而确定待测抗原或抗体的浓度。化学发光免疫分析具有分析方法简便、灵敏度高、线性范围宽、标记物有效期长、无放射性危害、可实现全自动化等优点，越来越受到人们的广泛关注。与各种技术如流动注射技术、高效液相色谱、毛细管电泳、电子偶合显像系统等广泛结合，化学发光免疫分析的应用范围得到了更进一步的发展。目前，化学发光免疫分析的发展趋势在于合成新的发光标记物、研究出更灵敏、发光量子产率更高的化学发光反应。化学发光免疫成像分析就是目前最受关注的化学发光免疫检测的方法之一，它是化学发光免疫分析与成像技术的联用，在实际应用中不但具有化学发光免疫分析的优点，同时也具有成像分析高通量，能多组分同时测定的优势。研究报告由四部分组成。第一个部分是从几种常见荧光探针（如丁基罗丹明、罗丹明B、罗丹明6G、四甲基异硫氰酸罗丹明B、荧光素、异硫氰酸荧光素等）的化学发光成像分析出发，建立了可以分析多种荧光物质的过氧化草酸酯-过氧化氢（H₂O₂）-咪唑-荧光物质化学发光体系。实验表明，利用过氧化草酸酯（TCPO）化学发光体系，对荧光探针的化学发光成像分析均具有高灵敏度、高通量、线性范围宽和检出限低等优势，比相同条件下荧光成像的检出限低一个数量级。第二个部分研究了多种辣根过氧化物酶催化反应产物的化学发光成像分析，实验将辣根过氧化物酶催化荧光反应与TCPO-H₂O₂-咪唑-荧光物质化学发光体系相结合，实现了TCPO化学发光体系在成像分析中的应用，这些酶催化反应体系包括HRP催化的还原型罗丹明B、酪氨酸、邻苯二胺（OPD）、3-（4-羟苯基）丙酸（PHPPA）等。利用酶催化化学发光成像，对HRP的定量分析既具有酶反应专一性好的优点，同时也具有良好的线性范围和检出限，这为TCPO化学发光体系用于免疫分析奠定了基础。第三、四部分是以酶催化荧光反应为纽带，将酶联免疫吸附法（ELISA）与TCPO化学发光成像分析相结合，建立了正常人血清样品中的α-干扰素（α-IFN）和癌胚抗原（CEA）定量分析的酶催化荧光化学发光免疫成像分析法。采用双抗体夹心法，在已包被有抗体的96孔酶标板中加入抗原标准品或待测样本，使之与吸附在固相载体上的特异性抗体反应，孵育后洗涤除去未反应的样品；加入酶（HRP）标记的特异性抗体与抗原反应；彻底洗涤后加入荧光底物和氧化剂过氧化氢，在37℃水浴中孵育一定时间后，取一定量的酶催化反应混合物质，利用TCPO-H₂O₂-咪唑-荧光物质化学发光体系对其进行化学发光成像分析，通过检测化学发光信号实现对抗原和待测样品的定量分析。此方法已经成功用于定量分析正常人血清中α-干扰素和癌胚抗原含量，在最优化条件下，发光强度值与α-IFN和CEA分别在1.3-156.0pg／ml（R²=0.9991）和1.0-128ng／ml（R²=0.998）范围内呈良好的线性关系，检出限分别为0.8pg／ml和0.5ng／ml，对25pg／mlα-IFN和32ng／mlCEA作9次平行测定的相对标准偏差分别为4.7％和4.2％。通过实验结果对照显示，酶催化荧光化学发光成像分析结果与标准的比色法诊断结果基本一致。实验结果表明：化学发光免疫成像分析法具有高通量、多元化检测、灵敏度高的特点，因此，用化学发光免疫成像分析法对某些疾病的超痕量临床诊断是可行的。更多还原

【Abstract】 This thesis consists of two parts.In the first part, a review about chemiluminescence immunoassay（CLIA）, the basicprinciples and new technologies of CLIA, the types and application ofchemiluminescence immunoassay imaging were summarized. CLIA is that usingchemiluminescence（CL） species or an enzyme as a label to study the reaction of antigenand antibody, and we can determine the CL intensity by affiliating the oxidant orenzyme to the chemiluminescence reaction after immunoassay reaction. In this way, theconcentrations of samples can be gained according to the CL intensity. Due to the mainadvantages of simple, high sensitivity, wide dynamic range, long activity of the label,non-radiation and automatic control, CLIA has raised worldwide interest. Combined tomany analytical methods such as flow injection, HPLC, capillary electrophoresis, andCCD （charge-coupled device） camera imaging, the application of CLIA could obtaindevelopment greatly. The current developments of CLIA are that they should synthesizenew labels in immunoassay analysis, develop new chemiluminescent systems that havemore sensitive and higher quantum yield.Combined CCD imaging technology to CLIA, Chemiluminescent immunoassayimaging-has the disadvantages of CLIA, and also has the advantages of high throughputand simultaneity detection of imaging device. It has been paid more and more attentionworldwide.The research part includes four sections. In the first section, a sensitive, simple andhigh throughput method has been used for quantitative analysis of several availablefluorescent probes （butyl rhodamine, rhodamine B, rhodamine 6G, tetramethylrhodamine isothiocyanate, fluorescein and fluorescein isothiocyanate and so on）, and anew chemiluminescent system, bis（2, 4, 6-trichlorophenyl） oxalate （TCPO）-hydrogenperoxide （H₂O₂）-glyoxaline-fluorescent substance, has been used in this research. Theresults showed that TCPO Chemiluminescence imaging analysis has the advantage ofsensitivity of chemiluminescence and high throughput of imaging. It had good linearand the detection limit was 10^-11 mol/1. In the second section, horseradish peroxidase（HRP）-catalyzed fluorescent reaction has been combined to TCPO-H₂O₂-glyoxaline-fluorescent substance chemiluminescence imaging analysis, and these enzymed-catalyzed reactions contain HRP catalyze tyramine, o-phenylenediamine（OPD）, 3-（4-hydroxyphenyl）-acetic acid（PHPPA） and the reduced forms of rhodamineB. Based on the advantages of enzymed-catalyzed reaction and chemiluminescenceimaging analysis, the quantitative analysis of HRP could be achieved by determinationof chemiluminescence intensity, and it had good linear and the determination limit was10^-14 g/ml. The investigation provides a possibility of using TCPO chemiluminescencesystem for immunoassay. Based on enzymed-linked immunosorbent（ELISA）, andHRP-catalyzed fluorescent reaction, TCPO chemiluminescence imaging analysis hasbeen used for determination of interferon alpha（α-IFN） and carcinoembryonic antigen（CEA） in fresh human serum samples in the third and forth section. A typical "sandwichtype" immunoassay was used. The antigen standard or samples were added into the96-well plates coated with antibody. After incubation in 37℃, washing steps followed,and the HRP-conjugated anti-α-IFN monoclonal antibody was added to react withantigen. After incubation and washing steps, the fluorogenic substrate and oxidant wereadded into the microtitre, then an incubation steps followed. After addition of mixture,TCPO chemiluminescence substrates were added to determine CL intensity. TCPOchemiluminescence imaging measurements have been successfully used for analysis ofthe content ofα-IFN and CEA in human serum samples. Under the optimum conditions,the CL intensity was proportional with the concentration ofα-IFN and CEA in the rangeof 1.3 to156.0pg/ml（R²=0.9991） and 1.0 to 128.0ng/ml（R²=0.998）, respectively. Thedetection limits were 0.8pg/ml and 0.5ng/ml（3σ）, respectively. The relative standarddeviation（RSD） for nine parallel measurements of 25pg/mlα-IFN and CEA 32ng/mlwere 4.7% and 4.2%, respectively. Compared with ELISA, the main advantages of thisnovel method is sensitive, simple, rapid, high throughput and simultaneous detection.The results indicate that the proposed CLIA method for the determination of somesubstance is feasible in clinical diagnoses.更多还原