發展快速檢測之蛋白質晶片及其應用

Abstract

本研究目的就是結合晶片的技術及免疫分析的方法，製作出一套可以快速定性及定量的蛋白質免疫晶片系統。它的優點是製作過程簡單，材料取得容易而且花費便宜，大幅縮短傳統生化檢測所需耗費的時間。此技術是利用微陣列機將所需要的檢測的抗原或抗體排印在特殊材質上，檢測時只需要微量血清樣本即可以加以定量人體血液中所含抗原或抗體的量。為了驗證我們所發展之新技術，我們以牛奶中一個特殊蛋白質β-lactoglobulin以及它的專一性抗體當作模組，用來測試晶片的實用性。經由測驗的結果發現，我們已經可以去除掉大部分雜訊的干擾並進行精確的定量，且將此新技術應用在酪農業上面，即可發展成快速判定的指標，用以檢測牛乳是否在不當的加熱滅菌過程中而導致營養成分流失。另外應用在醫學上面，我們也可以快速判定人體血清中apolipoprotein B (Apo B) 所含濃度，根據研究統計指出，測量Apo B比起只測量總血清膽固醇的含量，在預測心血管疾病的風險中具有更高的可信度。如果能進一步將檢測範圍延伸到測量Apo A-I的含量，並同時將Apo B及Apo A-I的比值當作心臟病病人的危險評估指標，即可達到早期發現早期治療而避免罹患心臟病的風險。以本研究成果為例，我們只需要25 μl反應試劑的體積，整個反應過程只要5分鐘就可以得到結果。由實驗結果顯示，本研究所發展的蛋白質免疫晶片，具備有高度的敏感性可以應用於各種生化檢驗上面，並達到精準及快速的結果。

A novel high-throughput microarray protein assay has innovatively been developed. This assay reserved superior advantages to the conventional immunoassays such as enzyme-linked immunosorbent assays (ELISA), enhanced chemiluminescence (ECL) and other high-throughput of microarrays. The array is based on it robotic printing technology. As such method can quantitate human sera antibodies or antigens in trace amounts. In our preliminary study, we determined the interactions between β-lactoglobulin and its monoclonal antibody. The method gave quantitative molecular-counting with an excellent signal without noise within a broad dynamic range, while retaining the realistic automation capability. Increased sensitivity and dynamic range were compared to currently array detection methods such as fluorescence, resulting in no signal bleaching, affording high reproducibility and compatibility with miniaturization. With this new method, apolipoprotein B (apoB) and apolipoprotein A-I (apoA-I) in human sera could be easily determined. Several studies have demonstrated that apoB in atherogenic particles—mainly LDL and VLDL; and apoA-I in anti-atherogenic particles—HDL, could provide a prediction of risk of coronary artery disease. In this study, only about 25 μL of diluted serum was required for completing this assay without any expensive laboratory equipment. Moreover, this assay had a simple visual end-point, and could be easily conducted in home within 5 minutes. Based on its sensitivity and specificity, this microarray assay would be useful in detecting variable pathogens presented in human sera. In conclusion, antibody microarray we developed was suitable for clinical diagnosis of infectious diseases.