【作者】 吴争鸣；

【导师】 吴朝阳；

【作者基本信息】 湖南大学 ， 分析化学， 2008， 硕士

【摘要】 发展高灵敏度的免疫分析技术一直以来是免疫分析领域研究的一个重要方面,它对于实现一些重要疾病标志物的检测有着至关重要的意义。荧光仪具有简便、灵敏、重现性好等优点。因此,开发新的荧光信号增强免疫分析技术无疑可以推动免疫分析的发展。为此,本文主要在高灵敏度的荧光免疫分析方法方面做了一些工作:1)提出了一种新的基于磁性纳米颗粒的荧光免疫检测方法。利用磁性纳米颗粒上的氨基固定C3抗原。研制了一种基于磁纳米颗粒作为生物载体,用于C3检测荧光免疫分析方法,将辣根过氧化物酶标记抗体(HRP-Ab)和磁性纳米颗粒上固定的C3( MNPs–C3)与待测C3发生竞争性免疫反应,HRP-Ab可以催化荧光底物3 ,3′,5 ,5′-四甲基联苯胺(TMB)发生淬灭,所以待测C3浓度可由底物溶液荧光猝灭的大小来判断。C3的检测范围为1 - 40 ng/mL,检测下限达到0.8 ng/mL,该方法满足血清样品中C3检测的要求。2)提出了一种基于磁性纳米颗粒的人胸苷激酶1(hTK1)荧光免疫分析方法。hTK1通过共价吸附到核壳结构的磁性颗粒表面,与样品中的hTK1共同竞争辣根过氧化物酶标记的抗体(HRP-Ab)然后以对羟基苯丙酸(HPPA)为底物,通过测定酶催化下荧光强度的增加来间接测定样品中抗原的浓度。应用于人血清中hTK的测定,在0.01 -1 ng/mL之间呈线性关系,检测限为0.008 ng/mL。3)提出了一种基于循环富集辣根过氧化物酶的用于检测人IgG的新型荧光酶免疫分析方法。脱硫生物素与亲合素的结合常数比较小,当其与亲合素结合后,在生理条件下即可被生物素取代,利用此取代反应可对辣根过氧化物酶进行循环富集。当用缓冲液代替抗原进行实验时,发现即使循环富集步骤达到七次其产生的背景信号也相当低,这种特性使得本方法能够检测到0.05 ng/mL人IgG。更多还原

【Abstract】 Developing of sensitive immunoassay technology is one of the important tasks in immunoassay, which is crucial for realizing the detection of some vital diseases. Fluorescent apparatus are simple, sensitive and good reproductivity. So it is undoubted that the study of new fluoroimmunoassay with signal enhancement can accelerate the development of immunoassay. Therefore, this research focuses on the sensitivity enhancement of fluoroimmunoassay. The detail contents are as follows:1) A fluorescence immunoassay based on magnetic nanoparticle immobilization was developed. C3 antigens were covalently immobilized through the amino groups on the core-shell type magnetic nanoparticles as a biological carrier. The horseradish peroxidase labled anti-C3 antibodies (HRP-Ab) were fixed onto the magnetic nanoparticles through the competitive immunoreactions with the bound C3 antigens (MNPs-C3) on the magnetic nanoparticles and the free C3 antigens in the sample solutions measurement. The fixed HRP-Ab can catalyze the fluorescent substrate 3, 3 ’, 5, 5’-Tetramethylbenzidine (TMB) to form a nonfluorescent product, so the C3 concentration can be sensitively measured according to the fluorescence quenching of the substrate solution. The C3 antigens can be linearly determined in the range of 1.0-40 ng/mL and the detection limit reaches 0.008 ng/mL.2) A fluorescence enhancing immunoassay for hTK1, using magnetic nanoparticles (MNPs) as the biomolecular immobilization mediator was developed. The horseradish peroxidase labeled hTK1 monoclonal antibody (HRP-Ab) competitively reacted with the free hTK1 in the detection solution and the bound hTK1 on MNPs. After separated by a magnetic field, the bound HRP-Ab on MNPs catalyzed the oxidization of 3-(p-hydroxyphenyl)-propionic acid (HPPA) to form a fluorescent dimmer product, 2,2’-dihydroxybiphenyl-5,5’-dipropionic acid, leading to an enhanced fluorescent signal at 407 nm which was inversely proportional to the hTK1 concentration. The hTK1 can be linearly determined in the range of 0.01-1 ng/mL and the detection limit reaches 0.008 ng/mL. Several live cancer patient and normal human serum samples were evaluated by the developed fluoroimmunoassay. This new immunoassay system can be extended to detect other biomarker molecules and has broad potential applications in clinical disease diagnosis.3) A new fluorescent enzyme immunoassay based on cyclic accumulation of horseradish peroxidase (HRP) was proposed for human IgG. The dissociation reaction between dethiobiotin and avidin in the presence of biotin provided efficient way for the cyclic accumulation of HRP used for the final analytical quantification. The blank experiment without addition of the analyte showed an extremely low background even with seven accumulation cycles reached. The proposed method provides a possibility for the determination of down to 0.05 ng/mL human IgG..更多还原