| Abstract: | 骨質疏鬆症是高齡化社會的普遍性疾病，更是停經後婦女及老年人健康上的隱形殺手，骨質疏鬆產生的骨折會對個人、家庭及國家造成龐大的經濟負擔，因此骨質疏鬆症已成為重要的研究課題。在骨質疏鬆的檢測方法中，骨質代謝生化指標是測定血液和尿液中骨質生成和耗損的代謝產物，可提供骨質疾病的變化和骨質代謝速率的資料，並評估骨質流失的情況。在骨質代謝指標中，第一型膠原蛋白是骨骼中數量最多的膠原蛋白類型，其交聯鍵結的N端胜肽片段（NTx）是人體中的骨骼膠原蛋白降解後所產生的物質，所以NTx為具有較高的特異性及敏感性的生化指標。根據本實驗室先前的研究結果，以NTx 片段為藍圖所設計的一系列線狀胜肽中，我們發現線狀胜肽peptide 2 (P2)與商業套組 Osteomark®中的anti-NTx抗體具有較高的親和效力，因此決定與美國Biocheck 公司合作，以P2胜肽為抗原製備人類單株抗體，以期開發評估骨質流失之方法。最近，我們利用anti-P2抗體製備免疫親和性管柱，分離病患之尿液中具有專一性的胜肽或蛋白質，並應用MALDI-TOF MS 偵測其質譜訊號。但是此方法需經沖提及去鹽的步驟才能進行偵測，其過程非常費時且無法同時檢測多個樣品，也可能造成回收率的降低。因此，在本論文中，我們應用ㄧ個免疫檢測方法，將anti-P2的人類單株抗體與表面經過修飾的磁性奈米粒子結合作為一個有效的親和性探針，同時可濃縮及分離尿液中具有專一性的胜肽。將此親和性探針與基質譜輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)結合可以偵測人類尿液中對anti-P2的人類單株抗體有親和性的胜肽。經過單株抗體修飾的磁性奈米粒子不需要沖提及去鹽的步驟，即可直接以MALDI-TOF MS鑑定與抗體結合的胜肽。此方法具有簡單、快速且可同時偵測多個樣品之優點。本論文的研究結果有助於研發骨質流失之檢測方法，對於骨質疏鬆症的預防治療及診斷，有所助益。
Abstract Osteoporosis is a continuously growing problem in the ageing populations and occurs very often in postmenopausal women and aged people. Osteoporotic fractures-related cost for patient, their families, and the country is huge. Therefore, osteoporosis has become an important subject for researches. Among the biomarkers of bone resorption, type I collagen crosslinked N-telopeptides (NTx) are terminal metabolites specifically derived from bone collagen degradation. Type I collagen is the major collagen product synthesized by bone cells and represents more than 90% by weight of the non-mineral component of bone. Thus, the rate of cross-linked N-telopeptides (NTx) excretion in urine is regarded as a highly specific index of bone resorption, and it is sensitively suppressed in response to anti resorptive therapies. In our previous studies, we found that the epitopes for anti-NTx antibodies lie within linear peptide 2 (P2) by using commercial ELISA (Osteomark?) and surface plasma resonance (SPR) technology. Therefore, we started the collaboration with Dr. John Chen at BioCheck, Inc. (Foster City, California, U.S.A). By using the P2 peptide-conjugate as the antigen, human monoclonal anti-P2 antibodies were prepared by BioCheck, Inc.. Recently, we used anti-P2 antibody to prepare immunoaffinity columns in order to separate peptides or proteins in patients’ urine for characterization by MALDI-TOF MS. But this method need the elution and desalting processes, which are time-consuming, are not able to simultaneously detect multiple samples, and may cause lower recoveries. Therefore, in this thesis, we have applied an immunoassay using monoclonal anti-P2 antibody-conjugated surface-modified magnetic nanoparticles (MNPs) as an efficient affinity probe to simultaneously preconcentrate and isolate targeted peptide from urines. We combined this affinity probe with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to profile peptides in human urine. The anti-P2 antibody-conjugated MNPs could be used directly in MALDI-TOF MS for characterization of antibody-bound peptides without the elution and desalting processes. This method is simple, fast and can simultaneously detect multiple samples. Results of these studies provide useful information for the development of methods for monitoring bone loss, which are important for the prevention, treatment, and diagnosis of osteoporosis. |
