量子点技术用于鼻咽癌标志物EBNA1的标记及初步应用研究

发布时间：2018-10-15 15:51

【摘要】：目的：探讨605nm羧基水溶性量子点双抗原夹心法在鼻咽癌标志物EBV EBNA1抗体中的快速检测及初步定量研究。方法：采用605nm羧基水溶性量子点共价交联的方法标记EBNA1抗原,量子点双抗原夹心法结合免疫层析技术及酶联免疫(elisa)法检测血清标本中EBNA1,并对其进行荧光半定量测定,初步绘制出EBNA1抗体标准品浓度吸光度标准曲线。结果：采用605nm羧基水溶性量子点和鼻咽癌EBV EBNA1抗原在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)的作用下共价交联,合成量子点标记的EBV EBNA1抗原,通过UV-2550紫外-可见分光光度计(SHIMADZU)和LS-55型荧光分光光度计证实605nm羧基水溶性量子点(PEG)-605已成功标记到EBNA1抗原上。采用605nm羧基水溶性量子点EBNA1双抗原夹心法结合elisa技术检测60例鼻咽癌患者及30例正常人血清中EBV抗体,灵敏度为91.67%,特异度为86.67%,使用中山生物工程有限公司生产的EBNA1IgA酶联免疫诊断试剂盒测得灵敏度为91.67%,特异度为80.00%,两种方法比较结果无明显差异,前者特异度稍高,但前者方法步骤更为简便,无需再加入显色剂。采用605nm羧基水溶性量子点双抗原夹心法结合免疫层析技术对不同浓度的EBVEBNA1抗体标准品进行荧光检测,发现当浓度低于25ng/mL时,即观察不到荧光。表明本方法对EBVEBNA1抗体标准品的检测最低检测限浓度为25ng/mL。利用605nm羧基水溶性量子点双抗原夹心法结合免疫层析技术快速检测60例鼻咽癌患者及30例正常人血清中EBV抗体,检测其flisa荧光度值,检测时间仅需3-10min,而传统的ELISA检测方法步骤繁琐,需数小时,统计分析证明鼻咽癌血清组和正常成人血清组所测的吸光度值差异明显,有统计学意义。并初步绘制出EBNA1抗体标准品浓度吸光度标准曲线,曲线相关系数R2=0.997,说明有比较好的相关性,可以实现实现样品的浓度测定。本实验对鼻咽癌患者血清及正常成人血清flisa吸光度值进行ROC统计,确定Flisa荧光值诊断鼻咽癌的最佳临界值为0.680817。该点灵敏度91.70%,特异度90.00%, Youden指数0.817,达到了理想的实验结果,并统计分析可疑值范围是(1.000988,1.091343)。结论： 1)通过共价交联法已经实现羧基水溶性量子点与鼻咽癌EBNA1抗原的成功标记。 2)量子点双抗原夹心法结合elisa技术检测切实可行,检测结果与传统的酶联免疫法相符,且无需加入显色剂。 3)量子点双抗原夹心法结合免疫层析技术检测切实可行,比elisa检测技术更为快捷,检测过程仅需3～10min。 4)初步绘制出EBNA1抗体标准品浓度吸光度标准曲线,可以实现样品的浓度测定,ROC统计,确定Flisa荧光值诊断鼻咽癌的最佳临界值为0.680817。该点灵敏度91.70%,特异度90.00%,Youden指数0.817,并统计分析可疑值范围是(1.000988,1.091343)。图13幅,表6个,参考文献40篇
[Abstract]:Objective: to investigate the rapid detection and quantification of 605nm carboxyl soluble quantum dot sandwich method in nasopharyngeal carcinoma (NPC) marker EBV EBNA1 antibody. Methods: 605nm carboxyl water-soluble quantum dots were used to label EBNA1 antigen, and double antigen sandwich method combined with immunochromatography and enzyme-linked immunosorbent assay (elisa) were used to detect EBNA1, in serum samples. The absorbance standard curve of EBNA1 antibody standard concentration was preliminarily plotted. Results: 605nm carboxyl water-soluble quantum dots (QDs) and nasopharyngeal carcinoma (NPC) EBV EBNA1 antigens were covalently crosslinked with 1- (3-dimethylamino-propyl) -3-ethylcarbodiimide hydrochloride (EDC) to synthesize QDs labeled EBV EBNA1 antigen. UV-2550 UV-Vis spectrophotometer (SHIMADZU) and LS-55 fluorescence spectrophotometer confirmed that 605nm carboxyl water-soluble quantum dot (PEG) 605 was successfully labeled on EBNA1 antigen. 605nm carboxyl water-soluble quantum dot EBNA1 double antigen sandwich method and elisa technique were used to detect EBV antibody in serum of 60 patients with nasopharyngeal carcinoma and 30 normal controls. The sensitivity was 91.67 and the specificity was 86.67. The sensitivity and specificity of EBNA1IgA Elisa kit produced by Zhongshan Bioengineering Co., Ltd. were 91.67 and 80.000.There was no significant difference between the two methods. However, the former method is more simple and no longer need to add chromogenic agent. 605nm carboxyl water-soluble quantum-dot sandwich method and immunochromatographic technique were used to detect the fluorescence of EBVEBNA1 antibody standard samples with different concentrations. It was found that no fluorescence could be observed when the concentration was lower than 25ng/mL. The results showed that the minimum detection limit for EBVEBNA1 antibody was 25 ng / mL. The EBV antibody in serum of 60 patients with nasopharyngeal carcinoma and 30 normal controls was detected by 605nm carboxyl water-soluble quantum dot sandwich method and immunochromatographic technique. The fluorescence value of flisa was measured in the sera of 60 patients with nasopharyngeal carcinoma and 30 normal controls. The detection time was only 3-10 mins, while the traditional ELISA detection method was tedious and needed several hours. Statistical analysis showed that the absorbance values of NPC serum group and normal adult serum group were significantly different and had statistical significance. The standard curve of concentration absorbance of EBNA1 antibody was drawn, and the correlation coefficient of the curve was 0.997, which indicated that there was a good correlation and the concentration of the sample could be determined. The absorbance of flisa in serum of NPC patients and normal adults was analyzed by ROC. The best critical value of Flisa fluorescence value for diagnosing nasopharyngeal carcinoma was 0.680817. The sensitivity, specificity and Youden index of this point were 91.70 and 0.817, respectively, and the range of suspicious values was (1.0009888) 1.091343. Conclusion: 1) the successful labeling of carboxyl water-soluble quantum dots with EBNA1 antigen of nasopharyngeal carcinoma has been realized by covalent crosslinking. 2) double antigen sandwich method of quantum dots combined with elisa technique is feasible. The results are consistent with the traditional enzyme linked immunosorbent assay (Elisa) and do not need to be added to the chromogenic reagent. 3) Quantum Dot double Antigen Sandwich method combined with immunochromatographic technique is feasible and faster than elisa technique. The standard curve of standard concentration of EBNA1 antibody can be drawn. The concentration of EBNA1 antibody can be determined by ROC statistics. The best critical value of Flisa fluorescence value for diagnosis of nasopharyngeal carcinoma is 0.680817. The sensitivity of this point is 91.70 and the specificity is 90.000.Youden 's index is 0.817, and the range of suspicious value is (1.0009881.091343). 13 figures, 6 tables, 40 references
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.63

【参考文献】