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標題: Optimization of protein/reactive dye reaction and it''s immunochromatographic application
作者: 王信仁
Wang, Hsin-Jen
關鍵字: immunochromatographic test;免疫層析檢測;reactive dye;human serum albumin;HSA;反應性染料;人血清白蛋白
出版社: 化學工程學系
Immuochromatographic test (ICT) has been widely used to detect antibody or antigen in agricultural, environmental and clinical tests as qualitative or/and qualitative tools for on-site and one-step testing. Application of ICT in clinical diagnostics greatly shortens the turn-around time in gaining clinical test results, which benefits the patients by its point-of-care testings performance. However, when dealing with multi-analytes assay, presently available ICT exhibits limitations from both qualitative and quantitative concern. Dye immunochromatographic test (DICT) which can be used in multi-colored test lines on test strips offers the advantage of easier distinction in diseases triage and wider analyte concentration ranges in quantitative as compared to the conventional one-colored ICT. In this study, we attempt to dye the antibody with mutiple colors for appling in DICT.
In this research, antibody against human serum albumin (anti-HSA) was colored by reacting with reactive dyes such as Procion MX and Unicion ES series. Two reactive dyes, Procion blue MX-7RX and Unicion red ES-2B, were selected for further dyeing process in consideration of their low non-specific affinity. The optimal dyeing conditions were found as follows; pH 11.4, 35.7℃, molar ratio 188 (mole dye/mole Ab), 45.6 minutes for Procion blue MX-7RX and pH 10.9, 40.1℃, molar ratio 54.6 (mole dye/mole Ab), 41.7 minutes for Unicion red ES-2B respectively. The non-specific binding during testing was successfully removed by adding casein and Tween-20 in the testing solution. Taking the HSA competition assay as examples, the dyed anti-HSAs measured HSA with the linear ranges from 100 to 3.12 µg/ml and from 12.5 to 1.56 µg/ml for Procion blue MX-7RX and Unicion red ES-2B, respectively.

免疫層析檢測(immunochormatiographic test, ICT)是利用抗體或抗原接上標誌物,以抗體與抗原間的免疫親和力做為依據的一種檢測方法,單一步驟即可完成檢測之優點,可應用於日常生活中的臨床診斷、污染偵測、衛生保健、農業及農業加工。顯然,目前多屬於定性分析上的應用,但是由於現場診斷(point-of-care testing, POCT)需要,免疫層析定量檢測亦成為研究者追求的目標。免疫層析檢測定量分析及多重待測物試紙開發之關鍵,在於多種色彩標誌物之開發。在本論文中,以不同顏色之染料作為標誌物,進行抗體染色反應,並將其應用於免疫層析檢測,此方式稱為染料免疫層析檢測(Dye immunochromatiographic test, DICT)。以多株抗體(polyclonal anti-HSA)及抗原(HSA)為檢測模式蛋白,探討反應性染料(PROCION BLUE MX-7RX及UNICION RED ES-2B)與多株抗體反應後,具最佳顯色強度之抗體染色條件,及染料分子與抗體及抗原間的非特異性吸附,並進一步探討染料免疫層析檢測在定量免疫分析上之應用。
結果顯示PROCION BLUE MX-7RX及UNICION RED ES-2B染料染色抗體,可加入casein及Tween-20,來解決非特異性吸附之問題。PROCION BLUE MX-7RX及UNICION RED ES-2B染料染色anti-HSA,最適化條件分別為pH 11.4、35.7℃、染料與抗體莫耳比188 (mole dye/mole Ab)及45.6分鐘;pH 10.9、40.1℃、染料與抗體莫耳比54.6 (mole dye/mole Ab)及41.7分鐘。PROCION BLUE MX-7RX及UNICION RED ES-2B染料染色抗體,以競爭性方式檢測抗原,抗原檢測線性範圍分別為,100~3.12 µg/ml及12.5~1.56 µg/ml。
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