免疫層析檢驗法在登革病毒檢測上之應用及開發

Abstract

Application of Immunochromatographic test on detection of dengue virus is helpful for the survey in the field and disease precaution. The development of detection of dengue virus is the main target of this master thesis. The following virus were used to assess the virus specificity and sensitivity of the strip test: dengue virus type Ⅰ、Ⅱ、Ⅲ、Ⅳ. Factors that affect the intensity of the testing result are investigated. The results show positive correlation between the appearance intensity and three factors: the concentration of immobilized antibody, the antigen concentration of sample solution, and the amount of protein to colloidal gold conjugate. The optimum pH and antibody concentration affect the quality of protein to colloidal gold conjugate. The optimum pH value is 8.2, and the optimum protein concentration is experientially adjusted, generally, more amount of protein the conjugate is more stable. The strip test result shows specific to dengue virus type Ⅱ. No cross-reactions were observed when samples of dengue virus Ⅰ、Ⅲ、Ⅳ type were tested by strip test.

開發登革病毒之快速免疫層析檢測法，將有助於田間調查與疾病預警的工作。本論文主要以開發檢測登革病毒的免疫層析試紙法為目標，了解與修正各種與顯色強度有關的因子，並且檢驗層析試紙對於登革病毒的特異性與靈敏度，以應用於登革病毒的檢測上。實驗結果，檢測時的顯色強度與固定化的抗體量、檢體的抗原量以及加入的標記物量有正相關，同時亦與標記物的品質有關；而製備標誌物時溶液的pH值、標誌特異性蛋白質的量與製得標誌物品質有關，最適的pH值為等電點附近，使用的登革抗體吸附膠體金的最適酸鹼值選擇pH 8.2。最適的蛋白質量則需要調整至足夠穩定吸附的量，加入的蛋白質量愈多製備的標誌物愈穩定。利用登革二型病毒單株抗體製作出的層析試紙特異性檢測，結果能夠專一的檢測到登革2型病毒，檢驗登革1、3、4型病毒則無顯色，這表示此檢測方法對登革2型病毒有特異性。本研究顯示免疫層析試紙法應用於登革病毒的快速檢測極具潛力。