基于铕离子荧光微球的降钙素原快速定量检测方法的建立

发布时间：2018-06-02 23:58

本文选题：降钙素原 + 铕离子荧光微球　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景:降钙素原(Procalcitonin,PCT)是诊断和监测细菌性炎症感染的一个重要临床诊断标志物,在全身炎症综合反应征和脓毒症患者的治疗和监测中具有极大的应用价值。目前临床上PCT有多种检测方法:胶体金层析检测灵敏度低、特异性不强;酶联免疫检测耗时耗力,操作繁琐;均相荧光检测也存在着操作时间长、检测成本高的缺点。目的:本研究用铕离子荧光微球作为抗体标记物,旨在建立一种快速、高灵敏度的降钙素原免疫层析方法,文中对建立的层析方法进行优化并对其性能进行评价;同时本研究初步探讨了铕离子荧光微球在磁分离均相免疫检测中的应用。方法:将PCT单克隆抗体(检测抗体)和鸡Ig Y标记上铕离子荧光微球(Europium(III)chelated microparticle,简称Eu-CM),然后处理在荧光结合垫上;另一株PCT单克隆抗体(包被抗体)包被于硝酸纤维素膜的检测线T位置;山羊抗鸡Ig Y抗体包被于硝酸纤维素膜质控线C位置,荧光垫和硝酸纤维素膜组装组成免疫层析试剂条。血清中的降钙素原与Eu-CM标记的检测抗体特异性结合形成抗原抗体反应复合物,反应复合物沿着硝酸纤维素膜前移,在硝酸纤维素膜T位置形成包被抗体-PCT-检测抗体-Eu-CM复合物,在C线位置形成山羊抗鸡Ig Y-鸡Ig Y-Eu-CM复合物,通过免疫荧光检测仪检测T、C位置的荧光信号。用本研究研制的免疫层析试剂条检测PCT重组抗原,用Sigma Plot 12.5分析该方法的标准曲线,空白检测限,最低检测限和功能性灵敏度;检测60份临床样本,Deming’s线性回归法分析该方法与法国梅里埃全自动酶联免疫荧光分析系统的相关性。在磁分离均相系统中,以PCT单克隆抗体标记Eu-CM作为检测抗体,另一株PCT单克隆抗体标记生物素作为固相抗体,偶联亲和素的磁微球用来分离Eu-CM-检测抗体-PCT-固相抗体-生物素复合物,初步探索Eu-CM在磁分离均相免疫检测中检测PCT重组抗原的最低检测限。结果:本研究建立了一种基于Eu-CM的降钙素原免疫层析检测方法,方法的最佳检测时间为15 min,免疫层析方法中PCT标准品空白检测限为0.01 ng/ml,最低检测限为0.02 ng/ml,检测范围0.02-25 ng/ml,功能性检测灵敏度为0.05 ng/ml(CV10%)。同法国梅里埃全自动酶联免疫荧光分析系统相比,两种检测方法间的线性相关系数为0.9864(n=60),两种方法学具有高度的相关性。Eu-CM应用于磁分离均相免疫检测中,标准曲线匹配二次函数,函数方程为y=19170.12+75493.74*X+(-26.00)*X2(R2=0.9986),PCT重组抗原最低检测限为0.04 ng/ml。综上所述,本研究为临床检测血清中PCT提供一种快速、准确的免疫层析方法;同时本研究中的Eu-CM也可以应用于磁分离均相免疫检测中,其在PCT重组抗原的检测中有较高的检测灵敏度,将来有可能成为临床PCT检测的又一种高灵敏度的检测方法。
[Abstract]:Background: Procalcitonin (PCT) is an important clinical diagnostic marker for the diagnosis and monitoring of bacterial inflammatory infection. It is of great value in the treatment and monitoring of systemic inflammatory syndrome and sepsis. At present, there are a variety of clinical detection methods for PCT: colloidal gold chromatography detection sensitivity is low, specificity is not strong; enzyme-linked immunosorbent assay (Elisa) time-consuming and labor-consuming, complicated operation; homogeneous fluorescence detection also has the shortcomings of long operation time and high detection cost. Objective: to establish a rapid and highly sensitive immunochromatographic method for calcitonin with europium ion fluorescent microspheres as antibody marker. At the same time, the application of europium ion fluorescent microspheres in magnetic separation homogenous immunoassay was discussed. Methods: PCT monoclonal antibody (detection antibody) and chicken IgY were labeled with Europium II Ichelated microparticle (Eu-CMN), and then treated on fluorescent binding pad. Another PCT monoclonal antibody (coated antibody) was coated in the T position of the detection line of the nitrocellulose membrane, and the goat anti-chicken IgY antibody was coated in the C-position of the nitrocellulose membrane quality control line. The fluorescent pad and nitrocellulose membrane were assembled to form immunochromatographic reagents. The serum procalcitonin specifically binds to the Eu-CM labeled antibody to form antigen-antibody reaction complex, which moves forward along the nitrocellulose membrane and forms a coated antibody -PCT- test antibody -Eu-CM complex at the T position of the nitrocellulose membrane. Goat anti-chicken Ig Y-Eu-CM complex was formed at the C-line position, and the fluorescence signal of TG-Y- chicken Ig Y-Eu-CM was detected by immunofluorescence detector. The recombinant PCT antigen was detected by immunochromatographic reagent strip. The standard curve, blank detection limit, minimum detection limit and functional sensitivity of the method were analyzed by Sigma Plot 12.5. 60 clinical samples were detected by linear regression method to analyze the correlation between the method and the automatic enzyme-linked immunofluorescence (Elisa) system of Merier in France. In the magnetic separation homogenous system, Eu-CM was labeled with PCT monoclonal antibody and biotin was labeled with another PCT monoclonal antibody as solid phase antibody. Magnetic microspheres of conjugated avidin were used to isolate Eu-CM-detection antibody -PCT- solid phase antibody biotin complex, and to explore the minimum detection limit of Eu-CM for detection of PCT recombinant antigen in magnetic separation homogenous immunoassay. Results: a method of procalcitonin proimmunochromatography based on Eu-CM was established in this study. The best detection time was 15 mins, the blank detection limit of PCT standard product was 0.01 ng / ml, the lowest detection limit was 0.02 ng / ml, the detection range was 0.02-25 ng / ml, and the sensitivity of functional detection was 0.05ng / ml CV10g / ml. The linear correlation coefficient between the two detection methods is 0.9864 (0.9864). Eu-CM is used in magnetic separation homogeneous immunoassay, and the standard curve matches quadratic function, compared with the French Merriere automatic enzyme-linked immunofluorescence analysis system, and the linear correlation coefficient between the two methods is 0.9864. The minimum detection limit of PCT recombinant antigen was 0.04 ng / ml. In conclusion, this study provides a rapid and accurate immunochromatographic method for clinical detection of PCT in serum, and the Eu-CM in this study can also be used in magnetic separation homogeneous immunoassay. It has high sensitivity in the detection of PCT recombinant antigen, and may become another high sensitivity detection method for clinical PCT detection in the future.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6

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