【作者】 付艳；

【导师】 李隆弟；

【作者基本信息】 清华大学 ， 分析化学， 2003， 硕士

【摘要】 本文提出了将高灵敏度的固体基质室温磷光技术与免疫反应专属性相结合建立固体基质室温磷光免疫法（solid substrates room temperature phosphorescence immunoassay，SS-RTP-IA）。进而，本文分别以异硫氰酸四甲基罗丹明（TRITC）为标记物通过直接方式测定了人IgG，以异硫氰酸曙红（Eosin5-ITC）为标记物通过夹心方式，生物素亲合素桥方式测定了人补体3，从而在方法学上论证了SS－RTP－IA的有效性，而且实验结果显示了此方法的高灵敏性和特异性。1．论文以TRITC作标记物用SS-RTP-IA法的直接方式测定了羊IgG。对TRITC标记的兔抗羊抗体（RAGAb-TRITC）及其和羊免疫球蛋白抗原（G-IgG）反应后的免疫复合物的磷光性质的研究结果表明RAGAb-TRITC和免疫反应后的复合物保留了TRITC的磷光性质，(maxex/(maxem=558nm/700nm。在测定羊IgG时，磷光信号强度与测量的羊IgG的量成线性关系，本方法的检出限为0.372pg/spot，（点样量0.4μl，分子量以150000计时，检出限2.48amol/spot）。和ELISA方法进行比较，此方法显示了更高的灵敏度，更好的线性关系，更宽的线性范围。并且，实验中用此法直接测定了羊血清中的IgG，用标准加入法证实了此方法的准确性，并和ELISA法的结果相符合。2．论文把生物素亲合素桥技术与SS-RTP-IA相结合，以异硫氰酸曙红Eosin5-ITC为标记物测定了人补体3。Eosin5-ITC，Eosin5-ITC标记的羊抗人补体3抗体，在聚酰胺膜上以Pb(Ac)2为重原子微扰剂时它们的最佳激发和发射波长没有明显的差别(maxex/(maxem=535nm/678nm。为了进行比较，论文先用夹心方式对人补体3进行了测定，检出限为0.546pg/spot（点样量0.4μl，分子量以185000计时，检出限2.95amol/spot）。通过生物素亲合素桥夹心式SS－RTP－IA法测定人补体3的实验中，磷光信号强度与人补体3的量成线性关系，检出限为1.094pg/spot（点样量0.4μl，分子量以185000计时，检出限5.91amol/spot）。同时，论文还对20个人血清试样中的人补体3进行了测定，并和ELISA方法进行了比较，结果符合的很好，这个结果证明此免疫方法不仅用SS-RTP作为一种测量工具，而且还有很好的特异性和选择性。更多还原

【Abstract】 This thesis proposed to set up SS-RTP-IA (Solid Substrate Room Temperature Phosphorescence Immunoassay), which is the combination of highly sensitive solid substrate room temperature phosphorescence technology and the speciality of immunological reaction. Human IgG was detected with TRITC (tetramethylrhodamine B isothiocyanate) as label through direct mode, human complement3 (C3) was detected with Eosin5-ITC (Eosin5-isothiocyanate) as label through sandwich mode and biotin-avidin sandwich mode in this thesis. Therefore, in methodology the validity of SS-RTP-IA is demonstrated, and the experimental results show high sensitivity and specialty of this assay.1. SS-RTP-IA based on an antibody labeled with TRITC to detect goat IgG through the direct mode was described in this thesis. The SS-RTP properties of rabbit anti-goat antibody labeled with TRITC (RAGAb-TRITC) and its immune complex with goat-immunoglobulin (G-IgG) were studied on a polyamide membrane (PM). The results showed that RAGAb-TRITC can react specifically with G-IgG on a PM, and retain the excellent SS-RTP property of TRITC, (maxex/(maxem=558nm/700nm. The dependence of the phosphorescence intensity on the amount of antigen G-IgG (in a sample volume of 0.4μl) was linear. Compared with enzyme-linked immunosorbent assay (ELISA), this assay showed a lower detection limit (0.37pg per spot), a better linear relationship and a wider dynamic range (0.62-30pg). The method was applied directly to determination of G-IgG in goat serum and the results agreed well with those obtained by ELISA. 2. Biotin-avidin bridge technology and SS-RTP-IA was combined to form biotin-avidin-SS-RTP-IA (BA-SS-RTP-IA). BA-SS-RTP-IA based on an antibody labeled with Eosin5-ITC to detect human complement3 (C3) was described. The results showed that RAC3-Eosin5-ITC can react specifically<WP=4>with C3 on a PM, and remain the excellent SS-RTP property of Eosin5-ITC, (maxex/(maxem=535nm/678nm. At the same time, SS-RTP-IA to detect human complement3 through sandwich mode was described. The detection limit was 0.546pg/spot (with a sample volume of 0.4μl). In BA-SS-RTP-IA, the dependence of the phosphorescence intensity on the amount of antigen human complement3 was linear. The detection limit was 1.094pg/spot (with a sample volume of 0.4μl). Compared with ELISA, the concentration of human complement3 in 20 human sera was determined. The results agreed well with those obtained by ELISA.更多还原