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研究生:林建良
論文名稱:發展Gentamicin的酵素連結免疫吸附分析法(ELISA)
論文名稱(外文):Development of enzyme-liked immunosorbent assay(ELISA) for gentamicin
指導教授:茅繼良
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:gentamicin酵素連結免疫吸附分析法
相關次數:
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農畜產品中抗生素的殘留被認為是造成抗藥性細菌的主要原因之一,為避免消費者接觸到含有gentamicin殘留的農畜產品,本實驗目的是發展gentamicin的酵素連結免疫吸附分析法,提供藥物殘留濃度檢測之用。本實驗以EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide)作為連結物(linker),製備gentamicin和horseradish peroxidase (HRP)的酵素結合體,並以相同方法製備gentamicin和human serum albumin的結合體,免疫紐西蘭白兔生產可辨識gentamicin的抗血清,完成gentamicin的競爭型ELISA檢驗試劑,並在PBS、豬血清、豬尿液及鮮乳中建立標準曲線並進行各項分析。實驗結果所生產的抗血清以稀釋20,000倍,配合酵素結合體稀釋4,000倍為最佳。敏感度在PBS中為1.994 ng/ml;在豬血清中為1.356 ng/ml;在豬尿液中為1.176 ng/ml;在鮮乳中為6.412 ng/ml。交叉反應以sisomicin 45.44 ﹪最高,其餘均低於0.2 ﹪。平均回收率在豬血清中為91.23 ﹪(76.66-110.70 ﹪);在豬尿液中為114.29 ﹪(98.03-106.58 ﹪);在鮮乳中為107.59 ﹪(89.462-131.599 ﹪)。組內及組間分析平均變異係數分別是1.26 ﹪和3.54 ﹪。動物實驗結果亦證明可檢測投藥後的血清及尿液。顯示所發展的ELISA專一性高、穩定且定量結果準確;敏感度低於我國所規定的殘留容許量標準(0.1-0.4ppm),可實際運用於第一線藥物殘留檢測使用。

目錄 I
表次 III
圖次 III
中文摘要 1
英文摘要(Abstract) 2
第一章 前言 3
第二章 文獻探討 4
第一節 Gentamicin的化學特性及藥理作用 4
(一)化學特性 4
(二)藥理作用 4
(三)在各種動物的藥物動力學 10
第二節 臨床用藥 15
(一)Once-daily dosing 15
(二)Once-daily dosing的優點 19
(三)Once-daily dosing的臨床使用 21
第三節 獸醫臨床上的使用及經濟動物的殘留實驗 23
(一)在小動物的使用方面 23
(二)在馬的使用方面 24
(三)在經濟動物的使用及相關殘留探討 27
第四節 Gentamicin的各種檢測方法 32
(一)Bioassay 32
(二)Chromatography 32
第五節 Gentamicin的免疫分析法與應用 37
(一)Radioimmunoassay 37
(二)Enzyme immunoassay 37
(三)Fluorescence polarization immunoassay 38
(四)Particle concentration fluorescence immunoassay 39
(五)Capillary electrophoretic immunoassay 40
(六)在抗生素殘留檢測的應用 40
第三章 材料與方法 42
第一節 Gentamicin抗原的製備 42
第二節 抗體的生產 43
第三節 Gentamicin酵素結合體的製備 45
第四節 棋盤方格法決定抗血清與酵素結合體稀釋倍率 46
第五節 建立各種基質標準曲線 49
第六節 交叉反應測試(Cross-reactivity) 50
第七節 回收試驗(Recovery test) 52
第八節 精確度(Precision) 53
第九節 動物實驗 54
第四章 結果 55
第一節 棋盤方格法、抗血清與酵素結合體最佳稀釋倍率 55
第二節 各基質中標準曲線的建立與敏感度的計算 55
第三節 交叉反應(Cross-reactivity) 56
第四節 回收試驗(Recovery test) 57
第五節 精確度(Precision) 59
第六節 動物實驗 60
第五章 討論 66
附錄1 77
參考文獻 78
表次
表1. 棋盤方格法結果 56
表2. 各種aminoglycoside類藥物與gentamicin之交叉反應率 57
表3. 在豬血清中回收試驗結果 58
表4. 在豬尿液中回收試驗結果 58
表5. 在鮮乳中回收試驗結果 58
表6. 組內分析精確度(Intra-assay precision) 59
表7. 組間分析精確度(Inter-assay precision) 59
圖次
圖1. Gentamicin結構式 14
圖2. Gentamicin於PBS中之標準曲線 61
圖3. PBS中gentamicin標準曲線之直線區段直線迴歸 61
圖4. Gentamicin於豬血清基質中之標準曲線 62
圖5. 豬血清中gentamicin標準曲線之直線區段直線迴歸 62
圖6. Gentamicin於豬尿液基質中之標準曲線 63
圖7. 豬尿液中gentamicin標準曲線之直線區段直線迴歸 63
圖8. Gentamicin於鮮乳基質中之標準曲線 64
圖9. 鮮乳中gentamicin標準曲線之直線區段直線迴歸 64
圖10. A豬血清中gentamicin濃度配合時間變化圖 65
圖11. B豬血清中gentamicin濃度配合時間變化圖 65

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