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研究生:蘇翰農
論文名稱:發展Clenbuterol的酵素連結免疫吸附分析法(ELISA)
論文名稱(外文):Development of enzyme-linked immunosorbent assay (ELISA) for clenbuterol
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
論文頁數:80
中文關鍵詞:Clenbuterol酵素連結免疫吸附分析法
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clenbuterol為一具選擇性之β2致效劑,其作用之機制是與2接受體結合後,經連串訊號傳遞後,活化protein kinase以形成其藥理作用的產生;其臨床主要用途是作為支氣管擴張劑,增加黏液纖毛的清除率 (Dixon., 1992),但由於其更具有改善屠體品質 (Choo et al., 1992 ),降低飼料換肉率的效果,故現非法被使用作為生長促進劑之濫用。本實驗的目的是建立clenbuterol的酵素連結免疫吸附分析法 (ELISA)之檢測技術,並對於不同基質的樣本進行檢測。發展之初是先將clenbuterol衍生成為diazotized clenbuterol,再與人血清白蛋白 (HSA)形成抗原,經多次對兔子的免疫以獲得抗體;另外,與蕖草根過氧化氫酵素 (HRP)形成酵素結合體,之後,以棋盤方格法決定抗體與酵素結合體的最佳稀釋倍率,並以競爭型酵素連結免疫吸附分析法的原理建立標準曲線,確立clenbuterol的ELISA檢測技術。本實驗所生產的抗體稀釋倍率為8000倍,酵素結合體則為2000倍;在PBS與馬血清中標準曲線的直線區段落在0.5-50ng/mL,於馬尿液中則是落在0.1-50ng/mL處;交叉反應率中以salbutamol與terbutaline的5.469%與4.534%最高,其餘的內源性兒茶胺及其代謝物的交叉反應率皆低於0.1%;以結合百分率50% (5 ng/mL)觀察,分析內與分析間變異值於PBS、馬血清及馬尿液中皆不超過10%;動物模式試驗方面,在馬血清中的濃度變化上,於停藥後9-15小時達到最高值 (0.84ng/mL),而馬尿液中的濃度於停藥後3小時達到最高值 (97ng/mL),兩者皆於之後便迅速地下降,直至停藥後216小時所可測得的微量濃度,此驗證了所發展的ELISA分析法可實際應用於動物樣本如血清及尿液的檢測上,且不會受到樣本中其他物質的干擾,也不會受到內源性兒茶胺的影響,可提供作為一具有準確性及穩定性的檢測技術。

Clenbuterol is one kind of β2 selective agonists. It binds withβ2 receptors then via series of signal transductions activating protein kinase to make pharmalogical effect. Clenbuterol clinically uses as bronchodilators by enhancing the clearance of mucosal cilliary, but it improves the carcass and lowers the feed efficacy. Illegal abusement using as growth promoter is common now. An ELISA was developed for the detection of clenbuterol in diversity. The clenbuterol derived diazotized clenbuterol and was conjugated with HSA to produce antigen. Anticlenbuterol serum was obtained after several times. Enzyme conjugate was produced with horseradish peroxidase (HRP). Checkerboard method was used to determine optimal concentrations of antibodies and enzyme conjugate. A standard curve was established suggesting that it is a competitive immunoassay. Working antiserum dilution was 8000x, and 2000x for enzyme conjugate. The detectable range was 0.5-50ng/ml in PBS and horse serum, and 0.1-50ng/ml in urine. The cross reactivity with salbutamol and terbutaline was 5.469% and 4.534%, respectively. The clenbuterol antiserum cross-reacted with other endogenous catecholamines and their metabolites less than 0.1%. Intra- and interassay variations were less than 10% in PBS, horse urine and horse serum. Horse serum concentration 9-15 hours following clenbuterol administration was maximum (0.84ng/ml), and horse urine 3 hours after administration concentration was maximum (97ng/ml). The both values declined rapidly, and they remained detectable for 216 hours. Results show that a sensitive and simple ELISA was developed for detection in horse urine and serum without extraction.

目 錄
目 錄 I
致謝……………………………………………………………………. III
摘要 IV
Abstract V
表次 VIII
圖次 IX
緒言 1
第一章 文獻探討 3
第一節 clenbuterol 的基本性質: 3
第二節 Clenbuterol常見檢測方式的發展 22
第二章 材料與方法 32
第一節 Clenbuterol輔抗原的製備 32
第二節 抗體的生產 33
第三節 酵素結合體的製備 35
第四節 抗體與酵素結合體倍率之決定 36
第五節 標準曲線的建立 38
第六節 交叉反應率的測試 39
第七節 分析內與分析間變異值計算 40
第八節 動物模式試驗 41
第三章 結果 43
第一節 抗體與酵素結合體的最佳稀釋倍率 43
第二節 標準曲線的建立與敏感度的計算 44
第三節 交叉反應率 48
第四節 分析內與分析間的變異值 49
第五節 回收曲線的建立 51
第六節 動物模式試驗 53
第四章 討論 55
參考文獻 60

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