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研究生:陳秋妹
研究生(外文):Chiu-Mei Chen
論文名稱:本土化直接式皮質醇酵素免疫分析系統之建立
論文名稱(外文):Establishment of Direct Enzyme Immunoassay for Canine Serum Cortisol and Evaluation of Canine Adrenal Dynamic Test in Taiwan
指導教授:蘇璧伶郭應誠
指導教授(外文):Biling SuIng-Cherng Guo
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:88
中文關鍵詞:皮質醇直接式酵素免疫分析法單株抗體ACTH刺激試驗低劑量類脂醇抑制試驗
外文關鍵詞:cortisoldirect EIAmonoclonal antibodyACTH-stimulation testlow-dose dexamethasone suppression test
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本研究的目的為利用融合瘤技術製備一高專一性之抗皮質醇單株抗體,並藉此單株抗體建立一簡便而可信賴之血清皮質醇酵素免疫分析法,再利用此分析法測試台灣地區健康犬隻經ACTH刺激試驗及類脂醇抑制試驗之前後血中皮質醇含量之變化,以獲得本土健康犬隻ACTH刺激試驗及類脂醇抑制試驗之正常基準值;另外,進行4種不同劑量對ACTH刺激試驗作一劑量效應評估。
利用cortisol-21-acetate-3-CMO-BSA免疫小鼠後,在PEG幫助下,將經免疫的小鼠脾臟細胞與骨髓瘤細胞進行細胞融合,經過選拔及限數稀釋後,獲得能產生抗皮質醇單株抗體的單株融合瘤,再利用此抗體建立競爭型酵素免疫分析法。本抗皮質醇酵素免疫分析法之靈敏度可達0.1 ng/mL,其分析內及分析間變異係數分別為8.28 ±0.09 %及14.60 ±0.28 %,對於其他類固醇之交叉反應性普遍很低,而直接測試1~10 ng/mL皮質醇在犬血清樣品的回收率為77.8~128.7%,顯示本分析法具高靈敏度、精確性及準確性;另外,平行試驗的平行結果則指出,本分析法可直接檢測犬血清樣品中的皮質醇濃度。
利用此分析法測試進行ACTH刺激試驗及低劑量類脂醇抑制試驗,得到53隻健康犬ACTH刺激試驗之刺激前皮質醇血清濃度為0.85~14.63 ng/mL,刺激後為3.06~37.22 ng/mL;由此結果分析不同的影響因子,發現皮質醇受ACTH刺激之上升程度並不受到性別及年齡的影響,但可發現體型較大之犬隻其皮質醇上升程度相對的較體型較小者為低。另外在11隻健康犬低劑量類脂醇抑制試驗結果,初步建立其基準值。而在ACTH之劑量效應的比較,4種不同劑量之ACTH對腎上腺所造成的刺激反應,以150μg/dog即可達到250μg/dog相同的刺激效果,而100μg/dog與50μg/dog 的刺激效果相同,但卻較150μg/dog及50μg/dog組為低;從體型大小來看,大型犬於相同的劑量下,其刺激效果明顯的較體型小者為低。
To clinically examine adrenal function, a direct enzyme immunoassay (EIA) for canine serum cortisol was developed by using monoclonal antibody. The immunized mouse splenocytes were fused with myeloma NS-1 cells to form antibody-secreting hybridomas. Following selection of HAT medium, the hybridoma secreting anti-cortisol antibody was cloned. The monoclonal antibody with a titer of 104 fold was used to establish a competitive EIA for cortisol. The sensitivity of EIA was 0.1 ng/mL with intraassay and interassay coefficients of variation of 8.28 ±0.09 % and 14.60 ±0.28 %, respectively. The recovery rates for 1, 5, 10 ng/mL of cortisol in dog serum were 77.8%~128.7% with direct detection. The parallelism test revealed its reliability to directly detect the cortisol level in canine serum without extraction. In generall, this EIA showed low cross-reactivities with a variety of steroids. In ACTH stimulation tests, the cortisol levels from 53 healthy dogs are 0.85~14.63 ng/mL before tests and 3.06~37.22 ng/mL after tests. The sex and age did not significantly affect the cortisol levels in response to ACTH, but the large breeds (BW>25 kg) had lower cortisol response than the smaller breeds. The results from dose-response experiments of ACTH stimulation test indicate that the 4 doses of ACTH efficiently stimulates the adrenal glands, and the dose of 150 μg/dog showed similar stimulating effect to the dose of 250μg/dog.
目 錄
目錄………………………………………………………….…...…….….1
表次……………..…………………………………………….………….…3
圖次…………………………………………………………….………….…4
摘要………………………………………………………..….…..……….5
Abstract………….………………………………………….…..……..…7
第一章 緒言……………………………………………….….………..9
第二章 文獻探討……………………………………....…..….…..11
第一節 皮質醇之生理調節及其病生理學…………………………….11
一、皮質醇之生理意義……………………………………………..11
二、下視丘-腦垂腺-腎上腺皮質軸之生理調節……………………12
三、腎上腺皮質機能障礙之病生理學………………………….….12
第二節 皮質醇之分析系統……………………………………….……17
一、放射免疫分析法(Radioimmunoassay, RIA)………………….17
二、酵素免疫分析法(Enzyme immunoassay, EIA)…………….18
三、單株抗體與多株抗體之比較……………………………………18
四、抗皮質醇酵素免疫分析系統……………………………………18
第三節 犬腎上腺皮質機能之動態測試……………………………….19
一、ACTH刺激試驗(ACTH-stimulation test)…………..….…19
二、低劑量類脂醇抑制試驗(Low dose dexamethasone suppression test, LDDST)………………………………………………21
三、ACTH刺激試驗之劑量效應……………………………………..22
第三章 材料與方法…………………………………………………….23
第一節 皮質醇之酵素免疫分析系統……………………………..….27
一、皮質醇單株抗體之製備…………………………………………27
二、競爭型酵素免疫分析法之建立…………………………………34
三、皮質醇酵素免疫分析法之評估…………………………………36
第二節 犬腎上腺皮質機能之動態測試………………………….……38
一、健康犬ACTH刺激試驗與類酯醇抑制試驗…………….…...…38
二、健康犬ACTH刺激試驗之劑量效應……………………….…...40
第四章 結果…………………………………………………………….41
第一節 皮質醇酵素免疫分析系統…………………………………….41
一、皮質醇單株抗體之製備………………………………………..41
二、競爭型酵素免疫分析法之建立……………………………..…42
三、皮質醇酵素免疫分析法之評估………………………………..42
第二節 犬腎上腺機能動態測試之評估……………………………….50
一、健康犬ACTH刺激試驗……………………………………......50
二、健康犬類酯醇抑制試驗………………………………..………60
三、ACTH刺激試驗之劑量效應……………………………....……64
第五章 討論…………………………………………………………….67
第一節 皮質醇酵素免疫分析系統………………………….…………67
一、皮質醇單株抗體之製備…………………………………..……67
二、皮質醇酵素免疫分析法之評估………………………..……..68
第二節 犬腎上腺機能動態測試之評估……………………………….71
一、健康犬ACTH刺激試驗…………………………….….……....71
二、健康犬纇酯醇抑制試驗………………………………………..73
三、ACTH刺激試驗之劑量效應…………………………………....74
第六章 結論…………………………………………………………….76
第七章 參考文獻……………………………………………………….78
附錄………………………………………………………………………...87
第七章 參考文獻
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