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研究生:陳丘泓
研究生(外文):CHIU-HENG CHEN
論文名稱:應用固相胜合成法(SPPS)合成typeIcollagencross-linkedN-telopeptide的片斷並應用ELISA及SPRtechnology測試其與抗體間之作用
指導教授:劉英明龍鳳娣
指導教授(外文):Liou, Y.-MLung, F.-D
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:90
中文關鍵詞:骨質疏鬆症骨質密度NTXELISA
外文關鍵詞:NTXELISAOsteoporosisBone mass density
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中文摘要
骨質疏鬆症是臨床上最重要的疾病之一,更是停經後婦女及老年人健康上的隱形殺手,嚴重影響老年時的生活品質,骨質疏鬆症問題之嚴重性已逐漸獲得重視。早期偵測出高危險群婦女,給予女性荷爾蒙補充治療,對預防骨質流失的療效巳被肯定。骨質密度的檢查,目前最常用的檢查儀是雙能量X光骨質吸收測定儀。骨代謝的生化檢查,分為骨生成指標及骨耗損指標,其中以偵測尿液中type I 膠原蛋白交聯鍵結之N-telopeptide(NTX)為具有特異性及敏感性較高的方法,但利用此項檢查無法達到優良篩檢試劑的簡單和經濟實惠之要求。本研究目的乃利用ELISA和SPR分析NTX胜片斷與抗體間之交互作用並探討其應用於骨質流失監測之潛力,研究結果有助於未來研發具有特異性,敏感性,簡易性及安定性,且經濟性高的檢測方法,以便提前採取預防措施,對於骨質疏鬆症的診斷,預防及治療有很大的助益。
關鍵詞: 骨質疏鬆症,骨質密度,NTX,ELISA

Abstract
Osteoporosis is a disease characterized by low bone mass which occurred very often in postmenopausal women and aged people, and affected the quality of their life. To prevent osteoporosis in postmenopausal women giving hormone in early stage is useful. But for some postmenopausal women, the effect of hormone treatment were not significance. The dual-energy X-ray absorptiometing was used to determine bone mineral density . Urinary level of the cross-linked N-telopeptide of type I collagen (NTX) has been reported to be a sensitive and specific marker of bone resorption. This sensitive inhibition enzyme-linked immunosorbent assay (ELISA) was established on microtitre plates to quantify the NTX in human urine. However, it is not very quick, cheap and convenient for monitoring bone resorption. We plan to study the interaction between each peptide fragment of NTX and anti-NTX antibody using both ELISA and SPR-based assay, and investigate their application to monitor bone loss. Our results should provide important information for further development of assays to monitor the bone loss to treat and prevent the osteoporosis.
Keywords: Osteoporosis; Bone mass density ; NTX ; ELISA

目錄 頁數
中文摘要…………………………………………………...1
英文摘要…………………………………………………...2
目錄………………………………………………….….…..3
第一章 緒論………………………………………………..6
第二章 研究動機與目的…………………………………..10
第三章 原理………………………………………………..13
第一節 固相胜合成技術(SPPS,solid phase peptide synthesis)
………………………………….…...13
1.1 固相胜合成於樹脂上……………………….13
1.2 利用化學裂解方法(chemical cleavage)得到胜粗
產物…………………………………………….13
1.3 胜合成之反應試劑…………………………..15
1.3.1 去保護基試劑(deprotecting reagent)-20%……….15
1.3.2 耦合試劑(coupling reagent)…………………..16
1.3.3 裂解試劑(cleavage reagent)…………………..17
第二節 高效能液相層析法(HPLC, high-performance liquid
chromatography )………………………………….18
第三節 質譜儀鑑定-快速原子撞擊法(FAB-MS, fast atom bombardment
mass spectrophotometry)………... 20
第四節 蛋白質樣品的定量……………..…………………20
第五節 酵素免疫沉澱分析試驗(ELISA, enzyme-linked immunosorbent
assay)…………………………….23
第六節 表面漿體共振技術(SPR, surface plasmon resonance)
technology…………………………………………24
第四章 材料與方法…………………………………………26
第一節 固相胜合成技術…………………………………26
1.1 固相胜合成之材料…………………………26
1.2 固相胜合成之方法…………………………26
1.3 化學裂解之材料………………………………28
1.4 化學裂解之方法………………………………28
第二節 高效能液相層析法………………………………….30
2.1高效能液相層析法之材料…………………….30
2.2高效能液相層析法之方法…………………….30
第三節 冷凍真空乾燥系統(freeze drying system)………… 32
第四節 蛋白質樣品的定量………………………………….33
第五節 酵素免疫沉澱分析試驗..………………………….. 34
5.1酵素免疫沉澱分析試驗之材料……………….34
5.2 酵素免疫沉澱分析試驗之方法………………35
第六節 表面漿體共振技術………………………………….36
6.1 SPR之材料…………………………………….36
6.2 SPR之方法…………………………………….36
第五章 結果………………………………………………… 39
第一節 胜之製備、純化、鑑定及樣品的定量之結果…….39
第二節 酵素免疫沉澱分析試驗之結果……………………..41
第三節 表面漿體共振技術之結果……………………… 43
第六章 討論………………………………………………… 46
參考文獻…………………………………………………….. 50
附件………………………………………………………… 55

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