本研究方法以發展低成本、操作便利、省時的均相酵素免疫分析技術(homogenous enzyme immunoassay, EIA)為研究目標，有別以往的非均相酵素免疫分析(heterogeneous enzyme immunoassay)方法，需要繁複耗時的操作程序、昂貴的偵測儀器系統，或是使用不安全且危險性高的化學試劑；本研究使用葡萄糖-6-磷酸脫氫(glucose-6-phosphate dehydrogenase , G6PDH)作為均相酵素免疫分析法的報告酵(reporter enzyme)，其因抗體誘發造成活性改變的特性，使之被應用在 5-羥色氨酸的標記，5-羥色氨酸為血清素的前驅物，在人體中為影響神經傳導與腦功能的重要物質；利用均相免疫分析的原理，不須額外的分離程序，即可使用簡易的紫外光、可見光光譜儀，於數分鐘內偵測奈米濃度範圍的 5-羥色氨酸。在研究中，一開始我們會先進 5-羥色氨酸與報告酵素的生物交聯，並利用蛋白質電泳與西方墨點法來確定生物交聯的結果，再進一步探討最佳化的實驗條件，以不同參數來進行酵素的修飾(包括改變 5-羥色氨酸抗原與交聯劑的反應活化時間，以及與酵素生物交聯的反應時間與濃度等)，並探討酵素活性抑制率與最佳化實驗參數的關係，從中獲得最高之酵素活性抑制的結果。

The goal of this study is to develop a less expensive, more convenient, less time-consuming homogenous enzyme immunoassay. Compare to the traditional heterogeneous enzyme immunoassay which requires tedious and time-consuming procedures, expensive detection systems, or involving hazardous chemicals, homogeneous enzyme immunoassay technology utilizes a reporter enzyme, such as glucose-6-phosphate dehydrogenase (G6PDH), which has the characteristics of antibody-induced inhibition and the enzyme will be used as the label of 5-hydroxytryptophan (5-HTP) which is the precursor of serotonin (5-hydroxytryptamine, 5-HT) and plays an important role in neurotransmission and the brain function. Based on the principle of homogeneous enzyme immunoassay, no additional separation process is required during the immunoassay process. The assay signal can be detected using a regular spectrophotometer and the concentration of 5-HTP at nanomolar levels can be detected in a few minutes. In this study, we will first start using protein electrophoresis and Western blot to determine the results of bioconjugation cross-linking and then we will optimize the experimental conditions, such as the activation time for modifying the carboxyl groups of 5-HTP and regent concentrations or the reaction time during the bioconjugation process, finally obtain maximum inhibition of enzyme activity results.

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