Development of immunochips for rapid detection of dengue virus in clinical specimens

• Main Authors: Chih-Cheng Su, 蘇至誠
• Other Authors: Tzong-Zeng Wu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/21801694535529366781

碩士 === 國立東華大學 === 生物技術研究所 === 91 === The global prevalence of dengue fever has grown dramatically in recent years. Until now, it is endemic in more than 100 countries in the world and becomes a major international public health concern. Unfortunately, the flu-like clinical symptom of dengue fever is untypical and the detecting procedures currently used are cumbersome and time-consuming. This is unfavorable for early stage epidemiological control and effective medical treatment. In this study, a new detection system was developed based on the quartz crystal microbalance (QCM) immobilizing with two monoclonal antibodies that specifically against the dengue virus envelope protein (E-protein) and non-structural 1 protein (NS-1 protein), respectively. Three immobilizing methods, glutaraldehyde, carbodiimide and protein A, were used to prepare the immunochips, then the dengue viral cultivation liquid, simulating samples with blending dengue virus into human serum and clinical specimens were examined by those chips to determing the best manufacturing process of immunochip and the most appropriate analytical procedure which could be applied for routine fast screening in hospital. Because protein A could bind the Fc portion of antibody to “orientationally” immobilize antibody onto QCM surface, it was the best among those three immobilizing methods. Due to the complex composition of human serum, immunochip could only effectively quantify dengue viral antigens after 1000 folds dilution of untreated simulating sample. But if the cibacron blue F3GA — heat denature (CB-HD) method has been employed to treat the simulating specimen before analysis, the dilution folds could reduce from 1000 to 100 folds and the detection limit could lower to 1.73 μg/ml (E protein) and 0.74 μg/ml (NS-1 protein). In the results of testing of clinical specimen, “cocktail” immunochip - which has two antibodies immobilized simutaneously - showed significantly potential to discriminate most dengue-positive case from negative serum specimen. Althrough it couldn’t quantify both antigens separately, the higher signal level makes it more suitable for screening of suspected patients in early stage.