光學誘發抗體抗原配對分離之應用研究

Photoinduce Antibody-Antigen Complexes to Dissociate

林玉雯(Yu-Wen Lin)

指導教授 ： 曾繁根 ； 楊重熙 ； 錢景常

國立清華大學/原子科學院/工程與系統科學系/碩士(2006年)

摘要

傳統分離抗體抗原配對辦法為利用低酸鹼值之酸性溶液洗滌，但此方法容易因時間控制不當破壞抗體或抗原，亦無法使用在活體內。本實驗中藉由可被光誘發解離氫離子的分子o-Nitrobenzaldehyde以酸鹼緩衝液PBS配製成溶液,使其照射UV光時解離氫離子使溶液中酸鹼值下降，無照光後溶液中酸鹼值可立即回復中性狀態，型成由光控制酸鹼值跳躍開關的溶液。量測結果得知o-NBA溶液照光下溶液中酸鹼值可達3.74，此酸鹼值已可使用來分離抗體抗原配對。利用FRET方式檢測抗體抗原間相互關係，抗體上接有螢光染劑(QD)於螢光顯微鏡下可被觀察而抗原上接有螢光quencher，當抗體與抗原相互接合時，會因QD與quencher間距離極小導致螢光被吸收而消光，經實驗證實當抗體與抗原比例1:100時消光程度可達約95%。最後將抗體抗原配對環境流入o-NBA溶液並同時照射UV光源，觀測此時螢光回覆率來辨別抗體抗原分離情形，實驗結果得知o-NBA溶液對抗體抗原配對分離確實有其效果，最高可達到以pH=3酸性溶液分離效果的85.5%。

關鍵字

抗體抗原配對 ； 分離 ； 光學誘發

並列摘要

This research proposes a photochemical method to dissociate immune complexes, decrease the hurt to protein of traditional acid washing by extremely shot operation time. We used the well-known characteristic of o-NBA molecules that can be photoinduced proton releasing by 365nm irradiation and make pH jump in the solution to dissociate immune complexes. In order to prove our proposal, we set up a flowing system, fixed antibody which has fluorescence and antigen modified with quencher which can quench the fluorescence in it. Because the effective distant of quencher quenches fluorescence is very shot (~10nm), only when antigens binds to antibodies, the distant is shot enough to quench the fluorescence. We judgment if antibody and antigen bind or not by fluorescence was quenched or not. The fluorescent dye we chose was quantum dots because of its fluorescence won’t be influenced by the pH value changes in the environment. Base on our results of experiments this method is workable, and it got similar recovery ratio to the solution pH=3.

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