定量检测tPSA和fPSA荧光免疫层析方法的建立

发布时间：2018-03-15 00:18

  本文选题：荧光微球　切入点：免疫层析　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景前列腺特异性抗原(Prostate specific antigen,PSA)由前列腺上皮组织分泌,分子量为34KD,已广泛应用于前列腺癌的辅助诊断,免疫学方法检测形式有两种,即游离前列腺特异性抗原(free-PSA,f PSA)、与α1-抗糜蛋白结合的前列腺特异性抗原(PSA-ACT),通常所说的总前列腺抗原(t PSA)是f PSA和PSA-ACT的总和。国际上认为,PSA浓度小于4ng/m L是正常水平,前列腺癌风险随PSA浓度升高而增加,当PSA浓度在4~10ng/m L之间时被认为是诊断灰区,t PSA和f PSA占t PSA百分比(%f PSA)常用来提高诊断灰区的前列腺癌诊断效率。定量检测t PSA和f PSA的方法主要有:化学发光法、时间荧光免疫分辨法、电化学发光法等,这些方法虽然具备灵敏度高、定量准确、重复性和稳定性较好等优点,但是需要大型仪器,适合样本量多的大中型医院,不适合单个标本测定,从而限制了t PSA和f PSA检测项目在社区医院的开展。目前国内检测t PSA和f PSA的试剂盒中,缺少一种可以准确、快速定量,并且操作简便可实现床旁检测方法,荧光微球免疫层析技术可实现床旁检测,如能研究成功,可弥补国内t PSA、f PSA检测方法的缺憾。目的建立一种性能优良的定量检测t PSA及f PSA的方法,为t PSA及f PSA的检测提供一种新的技术和方法。方法应用t PSA及f PSA蛋白制备室内参考品;用荧光微球与抗体偶联,并利用中心提供的荧光免疫层析实验技术和平台对本实验条件进行多方面的研究和优化。通过分析试纸条的最低检出限、特异性、准确度、线性、精密性以及稳定性,对其性能进行研究。取已经过罗氏试剂盒(电化学发光法)定值的血清87份,用本实验建立的检测方法进行测定,比较两种检测方法的相关性。结果研究结果显示,本实验制备的t PSA及f PSA室内参考品在≤-20℃环境下至少能够稳定保存12个月,2-8℃环境下至少能够稳定保存4周;本试验制备的试纸条t PSA的最低检出限为0.08ng/m L,f PSA的最低检出限为0.06ng/m L;与CEA、AFP、CA125均无交叉反应;试纸条批内变异系数均小于10%,批间变异系数均小于15%,回收率在97.06%-103.11%之间;试纸条在2-8℃储存条件下至少可稳定存放6个月,37℃储存条件下可稳定存放10天;用本实验建立的荧光免疫层析试纸条与罗氏试剂盒(电化学发光法)进行相关性分析,检测t PSA的R2为0.9727,回归方程为Y=0.9372X+0.2626,检测f PSA的R2为0.9888,回归方程为Y=0.9981X-0.0537,两种检测方法具有良好的相关性。结论(1)制备出稳定的t PSA、f PSA室内参考品;(2)初步建立了同时检测t PSA、f PSA荧光层析定量检测方法。(3)制备了性能良好的t PSA、f PSA荧光层析定量检测试纸条。
[Abstract]:Background Prostate specific antigenase (PSAs) is secreted from the epithelium of the prostate with a molecular weight of 34kD. It has been widely used in the auxiliary diagnosis of prostate cancer. There are two kinds of immunological methods for detection of prostate cancer. That is, free prostate specific antigen free-PSAF PSAA, a prostate-specific antigen (PSA-ACTA) binding to 伪 1-antichyme protein, is the sum of f PSA and PSA-ACT. The risk of prostate cancer increases with the increase of PSA concentration. When the concentration of PSA is between 4 ~ 10 ng / mL, it is considered to be a diagnostic gray area t PSA and f PSA as a percentage of t PSA) is often used to improve the diagnostic efficiency of prostate cancer in the gray area. The main methods for quantitative detection of t PSA and f PSA are chemiluminescence method. Although these methods have the advantages of high sensitivity, accurate quantification, reproducibility and stability, they need large instruments and are suitable for large and medium-sized hospitals with a large sample size. T PSA and f PSA are not suitable for single specimen determination, which limits the development of t PSA and f PSA detection in community hospitals. At present, there is a lack of a kit for detecting t PSA and f PSA in China, which can be accurately and rapidly quantified. It is easy to operate and can be used for bedside detection. Fluorescence microsphere immunochromatography can be used to detect bedside, if it can be studied successfully, This method can make up for the shortcoming of domestic t PSA and f PSA detection methods. Objective to establish a method for quantitative detection of t PSA and f PSA with good performance. Methods the t PSA and f PSA proteins were used to prepare indoor reference materials, and fluorescent microspheres were used to couple with antibodies, and a new technique and method for the detection of t PSA and f PSA were provided. The experimental conditions of this experiment were studied and optimized by using the fluorescence immunochromatographic technique and platform provided by the center. The minimum detection limit, specificity, accuracy, linearity, precision and stability of the test strip were analyzed. 87 samples of serum which had passed the value of Roche kit (electrochemiluminescence method) were used to determine the correlation between the two methods. T PSA and f PSA reference materials prepared in this experiment can be kept stably for at least 12 months and 2 to 8 鈩，

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