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標題: 商業化染料於免疫層析檢測上之應用
Development of Immunochromatographic Tests with Commercial Dyes and Their Applications
作者: 王淑璟
Wang, Shwu-Jinng
關鍵字: Reactive dyes;反應性染料;Immunochromatographic test;Optimal reaction conditions;Response surface methodology;Disperse dyes;Enzyme-linked immunosorbent assay;Infectious bursal disease virus;Dextran;Microalbumin;Point of care testing;分散性染料;葡聚糖;免疫層析檢測;最適化反應條件;回應曲面法;酵素連結免疫吸附分析;傳染性華式囊病病毒;微量白蛋白;醫護地點檢測
出版社: 化學工程學系所
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本論文分成三部份,首先探討使用反應性染料(reactive dye)作為標誌物,於免疫層析檢測(immunochromatiographic test, ICT)之應用;接著選用葡聚糖(dextran)作為染色介質,以開發出顯色增強之染料免疫層析檢測系統;最後應用分散性染料(Disperse dye)作為顯色標誌物,並使用於華氏囊病病毒(infectious bursal disease virus, IBDV)抗體之檢測。
在本研究中,以氯化三聚氫酸系反應性染料PROCION BLUE MX-7RX染色抗體,以作為免疫檢測之標誌物,並將其應用於免疫層析檢測上。以多株抗體(polyclonal anti-HSA)及抗原(HSA)為檢測之模式蛋白質,探討抗體經由反應性染料進行染色反應後,其在免疫層析檢測試紙上之最佳顯色強度。結果顯示:在pH 11.4,35.7℃,染料與抗體莫耳比188 (mole dye/mole Ab),反應時間為45.6分鐘時;以 PROCION BLUE MX-7RX(PBM)染色抗體anti-HSA,可得到最佳顯色強度約為8738,且由結果分析可知,抗原與染色抗體間仍可維持良好之親合力。
由於染料可呈現不同顏色,因此染料免疫層析檢測具有同時檢測多重待測物的發展潛力。為進一步改良免疫層析檢測之顯色結果,本研究中將利用葡聚糖作為染色中間介質,結合反應性染料染色介質作為標誌物,探討其在染料免疫層析檢測上之應用。此染料染色介質之標誌物,利用間接染色方式結合反應性染料(PBM)於改質過之葡聚糖(PBM-modified dextran , PMD),並進一步探討抗體以此標誌物進行染色反應後,於免疫層析檢測試紙上之最佳顯色強度。結果顯示:葡萄糖單元與氯乙酸莫耳比1︰3 (glucose units/chloroacetic acid),葡聚糖分子量為10 kDa,反應時間10小時;利用染料染色改質後之葡聚糖,作為增強顯色之染料標誌物,以人血清白蛋白(human serum albumin, HSA)及其抗體(antiHSA)為檢體模式,可得免疫層析試紙上之最佳顯色強度約為29,898。對比與蛋白質直接染色法之結果,顯示此間接染色法可增強顯色強度達3.42倍。另外,應用在標誌抗體進行競爭型檢測,並以手提式顯色儀進行數據紀錄,檢測線性範圍為0 to 50 μg/ml,偵測極限為0.12 μg/ml。
第三部份的研究中,利用分散性染料(disperse dye, DADISPERSE NAVY BLUE SP)作為標誌物以檢測華式囊病病毒抗體。應用分散性染料開發之免疫層析檢測試紙進行反應條件探討,以測試線上之顯色強度為目標函數,結果顯示染料濃度吸光值為3 (λmax=587 nm),pH 7,50℃ 下反應10分鐘;利用分散性染料標誌抗體(rabbit anti-chicken),得到最佳顯色強度約為55,054。分散性染料之免疫層析試紙的定性分析中,檢測來自不同養雞場之雞隻血清樣品,藉由分析血清樣品之抗體力價,並與市售酵素連結免疫分析套組(FlockChek enzyme linked immunosorbent assay kits)進行比對,由數據結果顯示,染料免疫檢測試紙具有高度靈敏度及特異性。再與市售檢測套組進行定量結果探討,由分析試紙之顯色強度與市售檢測套組之抗體力價可知,其具有良好之線性關係(r2 = 0.9687)。為評估雞隻接種疫苗後,血清中是否已被誘發產生足夠的抗體力價,並利用層析檢測試紙及市售檢測套組,進一步分析不同雞群之血清樣品;經比對後發現,此兩種檢測方法在血清樣品群體的平均力價檢測上,具有良好的一致性結果。因此,以分散性染料作為標誌物之免疫層析檢測試紙,於定性及定量結果中均顯示其具有可信賴性、便宜及操作簡易等特性,相當適合開發於現場檢測之應用上。

This study was divided into three parts involving the feasibility of using reactive dyes as an immunoassay marker in immunochromatographic test (ICT) , an improved dye immunochromatographic test (DICT) using dextran as conjugate spacer, and the feasibility of using disperse dyes as a chromogenic marker for the detection of antibodies against infectious bursal disease virus.
For investigating the feasibility of using reactive dyes as an immunoassay marker, a dichlorine triazine dye, Procion Blue MX-7RX (PBM), was employed to stain the antibody against human serum albumin (anti-HSA). With the color intensity revealed in the ICT strip as the objective variables, the optimal dyeing conditions were found as follows: pH 11.4, temperature 35.7 ℃, molar ratio 188 (mol dye/mol antibody), and reaction time 45.6 min. The dyed-anti-HSA revealed a maximal color intensity of 8738 without apparent loss of antigen binding affinity.
An improved dye immunochromatographic test (DICT) using dextran as conjugate spacer loading dye molecules to enhance chromophor color intensity with the potential of simultaneous multi-colored assay was developed. To construct this new effective chromophor, a dyeing process was carried out by coupling a reactive dye PBM to modified dextran (MD). The optimal conjugate condition between PBM and MD was studied. It showed that under the optimized dyeing conditions, a molar ratio (glucose units/chloroacetic acid) of 1:3, dextran MW of 10 kDa, and a dyeing time of 10 h were obtained. The resulting dyed-dextran chromophor, used as both spacer and color intensifier, was further labeled to a model antibody, anti-human serum albumin (antiHSA), to build a PBM-dextran-antiHSA (PDA) conjugate. The PDA thus obtained generated the highest color intensity of 29,898 assayed by DICT strips and densitometer scanning. As compared to protein direct dyeing, this indirect dyeing protocol yielded a chromophor 3.42 times increase in color intensity calculated on equal molar HSA basis. A competitive DICT for determination of HSA using a handheld rapid-test scanner was carried out. A linear range between 0 to 50 μg/ml with a detection limit of 0.12 μg/ml was observed.
For investigating the feasibility of using disperse dyes as a chromogenic marker, a disperse dye, DADISPERSE NAVY BLUE SP, was selected in analyzing antibody against infectious bursal disease virus (anti-IBDV). With the color intensity revealed in the disperse dye ICT strip as the objective function, the optimal dyeing conditions were found as follows: dye concentration absorbance (at λmax = 587 nm) = 3, pH 7, 50oC, for 10 min. Under these conditions, the resultant dyed-antibody (rabbit anti-chicken) can produce an optimal color intensity reading of 55,054 on the strip. For performing qualitative immunoassay, chicken sera samples taken from different farms were used for the anti-IBDV titre assessment. The results of DICT strips showed very high sensitivity and specificity as compared to that analyzed by FlockChek enzyme linked immunosorbent assay (F-ELISA) kits. For quantitative immunoassay, it was found that the color intensity measured with DICT was linearly correlated to that of F-ELISA titre (r2 = 0.9687). Therefore, DICT was further applied to the detection of chicken anti-IBDV sera under vaccination in the farms. The average titres of the sampling groups exhibited a strong agreement to that of F-ELISA. Accordingly, the DICT method developed in this study, shown to be reliable, cheap and simple in both qualitative and quantitative immunoassays, is particularly suitable for point-of-need testing (PONT) in agricultural applications.
其他識別: U0005-0302201017200200
Appears in Collections:化學工程學系所

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