量子點技術(shù)用于鼻咽癌標(biāo)志物EBNA1的標(biāo)記及初步應(yīng)用研究
發(fā)布時間：2018-10-15 15:51
【摘要】：目的：探討605nm羧基水溶性量子點雙抗原夾心法在鼻咽癌標(biāo)志物EBV EBNA1抗體中的快速檢測及初步定量研究。 方法：采用605nm羧基水溶性量子點共價交聯(lián)的方法標(biāo)記EBNA1抗原,量子點雙抗原夾心法結(jié)合免疫層析技術(shù)及酶聯(lián)免疫(elisa)法檢測血清標(biāo)本中EBNA1,并對其進(jìn)行熒光半定量測定,初步繪制出EBNA1抗體標(biāo)準(zhǔn)品濃度吸光度標(biāo)準(zhǔn)曲線。 結(jié)果：采用605nm羧基水溶性量子點和鼻咽癌EBV EBNA1抗原在1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽(EDC)的作用下共價交聯(lián),合成量子點標(biāo)記的EBV EBNA1抗原,通過UV-2550紫外-可見分光光度計(SHIMADZU)和LS-55型熒光分光光度計證實605nm羧基水溶性量子點(PEG)-605已成功標(biāo)記到EBNA1抗原上。 采用605nm羧基水溶性量子點EBNA1雙抗原夾心法結(jié)合elisa技術(shù)檢測60例鼻咽癌患者及30例正常人血清中EBV抗體,靈敏度為91.67%,特異度為86.67%,使用中山生物工程有限公司生產(chǎn)的EBNA1IgA酶聯(lián)免疫診斷試劑盒測得靈敏度為91.67%,特異度為80.00%,兩種方法比較結(jié)果無明顯差異,前者特異度稍高,但前者方法步驟更為簡便,無需再加入顯色劑。 采用605nm羧基水溶性量子點雙抗原夾心法結(jié)合免疫層析技術(shù)對不同濃度的EBVEBNA1抗體標(biāo)準(zhǔn)品進(jìn)行熒光檢測,發(fā)現(xiàn)當(dāng)濃度低于25ng/mL時,即觀察不到熒光。表明本方法對EBVEBNA1抗體標(biāo)準(zhǔn)品的檢測最低檢測限濃度為25ng/mL。 利用605nm羧基水溶性量子點雙抗原夾心法結(jié)合免疫層析技術(shù)快速檢測60例鼻咽癌患者及30例正常人血清中EBV抗體,檢測其flisa熒光度值,檢測時間僅需3-10min,而傳統(tǒng)的ELISA檢測方法步驟繁瑣,需數(shù)小時,統(tǒng)計分析證明鼻咽癌血清組和正常成人血清組所測的吸光度值差異明顯,有統(tǒng)計學(xué)意義。并初步繪制出EBNA1抗體標(biāo)準(zhǔn)品濃度吸光度標(biāo)準(zhǔn)曲線,曲線相關(guān)系數(shù)R2=0.997,說明有比較好的相關(guān)性,可以實現(xiàn)實現(xiàn)樣品的濃度測定。本實驗對鼻咽癌患者血清及正常成人血清flisa吸光度值進(jìn)行ROC統(tǒng)計,確定Flisa熒光值診斷鼻咽癌的最佳臨界值為0.680817。該點靈敏度91.70%,特異度90.00%, Youden指數(shù)0.817,達(dá)到了理想的實驗結(jié)果,并統(tǒng)計分析可疑值范圍是(1.000988,1.091343)。 結(jié)論： 1)通過共價交聯(lián)法已經(jīng)實現(xiàn)羧基水溶性量子點與鼻咽癌EBNA1抗原的成功標(biāo)記。 2)量子點雙抗原夾心法結(jié)合elisa技術(shù)檢測切實可行,檢測結(jié)果與傳統(tǒng)的酶聯(lián)免疫法相符,且無需加入顯色劑。 3)量子點雙抗原夾心法結(jié)合免疫層析技術(shù)檢測切實可行,比elisa檢測技術(shù)更為快捷,檢測過程僅需3～10min。 4)初步繪制出EBNA1抗體標(biāo)準(zhǔn)品濃度吸光度標(biāo)準(zhǔn)曲線,可以實現(xiàn)樣品的濃度測定,ROC統(tǒng)計,確定Flisa熒光值診斷鼻咽癌的最佳臨界值為0.680817。該點靈敏度91.70%,特異度90.00%,Youden指數(shù)0.817,并統(tǒng)計分析可疑值范圍是(1.000988,1.091343)。 圖13幅,表6個,參考文獻(xiàn)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
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R739.63
本文編號：2273025
[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
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R739.63
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