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研究生:林怡祺
論文名稱:以酵素連結免疫吸附分析法檢測乳牛於乳房內注射cloxacillin後的牛奶與血清內cloxacillin之濃度
論文名稱(外文):Using enzyme-linked immunosorbent assay (ELISA) for the detection of cloxacillin concentrations in milk and serum after intramammary administration of cloxacillin to a cow
指導教授:陳鵬文陳鵬文引用關係
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:65
中文關鍵詞:cloxacillin酵素連結免疫吸附分析法牛奶
外文關鍵詞:cloxacillinenzyme-linked immunosorbent assaymilk
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  • 被引用被引用:1
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為了利用實用、簡單且方便的cloxacillin酵素連結免疫吸附分析法(ELISA)之檢測技術,以適用於牛奶樣品中藥物殘留之檢測,並期望此診斷試劑之敏感度能達到10 μg/kg以下。本實驗先製造cloxacillin與horseradish peroxidase(HRP)之結合體,並利用市售之抗cloxacillin抗體,以棋盤方格法來決定最佳的抗體與酵素結合體之稀釋倍率組合,結果顯示,以抗體稀釋1600倍和酵素結合體稀釋1600倍為最佳組合倍率。而本實驗所建立之競爭性ELISA可以在PBS和牛奶中進行檢測cloxacillin,其標準曲線之直線區域均落於5~1000 ng/ml之間,而敏感度分別為3.16 ng/ml和5.68 ng/ml。在交叉反應率方面,僅對oxacillin和dicloxacillin有交叉反應(分別為22.6%和55.3%),對於其他β-lactam類抗生素之交叉反應率則均小於1%。分析內和分析間變異值均小於10%,表示此ELISA在抗體辨識抗原以及操作的一致性上是穩定且可以信賴的。在動物試驗方面,乳牛乳房內注射三次乳房炎軟膏後,牛奶中cloxacillin的濃度於注射後2小時達最高濃度366,944 ng/ml,微量的濃度於第10天仍可以被檢測到,血清內cloxacillin的濃度於注射後2小時達最高濃度55.74 ng/ml,檢測到第12小時,顯示所使用之cloxacillin ELISA診斷試劑可實際應用於檢測上,而於鮮乳之市場調查中並未檢測出有超過10 ppb之cloxacillin殘留的情形。由本實驗所使用之cloxacillin ELISA診斷試劑,可用於檢測牛奶內cloxacillin之殘留,而不會受牛奶內其他物質的干擾,未來希望能自行製備抗cloxacillin之抗體,使本技術能更臻完善。

An useful、simple and convenient ELISA with a sensitivity of less than 10 μg/kg was used for the detection of cloxacillin in milk samples. The labeled antigen was produced by combining cloxacillin with horseradish peroxidase(HRP), while the anti-cloxacillin antibody was obtained from Biogenesis®. Checkerboard method was used to determine the optimal concentrations of antibody and enzyme conjugate. The working antibodies were diluted into 1600x, and the same with enzyme conjugates. Standard curves were obtained by the competitive immunoassay in PBS and in milk, respectively, and both the detectable ranges were from 5 to 1000 ng/ml. The detection limit in PBS and in milk was 3.16 ng/ml and 5.68 ng/ml, respectively. The cross-reactivity of oxacillin and dicloxacillin in this method was 22.6% and 55.3%, respectively, while it was less than 1% with other β-lactam antibiotics. Intra- and inter-assay variations in PBS and in milk were less than 10 %. Intramammary infusion of cloxacillin and ampicillin was given triply into the infected quarter in a cow with mastitis at 12 hours interval. The maximal concentration of cloxacillin in milk and in serum continued until 2 hours after treatment with 366,944 ng/ml and 55.74 ng/ml, respectively. Cloxacillin residue in milk and in serum was detectable for 10 days and 12 hours, respectively. The result of detecting all the different market milk showed that the cloxacillin residue was lower than 10 ng/ml. The ELISA kit used in this study was successfully applied to analyze cloxacillin in milk, and was not interfered by other antibiotics. Therefore, it needs a further study to produce the antibody of cloxacillin to complete this ELISA kit.

第一章 緒言---------------------------------------------------1
第二章 文獻探討
第一節 盤尼西林類抗生素----------------------------------------2
第二節 Cloxacillin運用於乳牛乳房炎之治療-----------------------7
第三節 檢測牛奶內Cloxacillin之殘留----------------------------13
第四節 牛奶內Cloxacillin之酵素連結免疫吸附分析法--------------26
第三章 材料與方法
第一節 酵素結合體之製備---------------------------------------33
第二節 棋盤方格法決定抗體與酵素結合體稀釋倍率-----------------34
第三節 標準曲線之建立-----------------------------------------37
第四節 交叉反應之測試-----------------------------------------39
第五節 分析內與分析間變異值之計算-----------------------------40
第六節 回收曲線與回收率之建立---------------------------------40
第七節 動物試驗與市售鮮乳之檢測-------------------------------40
第四章 結果
第一節 抗體與酵素結合體之最佳稀釋倍率-------------------------42
第二節 標準曲線之建立與敏感度之計算---------------------------45
第三節 交叉反應率---------------------------------------------48
第四節 分析內與分析間之變異值---------------------------------49
第五節 回收曲線之建立-----------------------------------------50
第六節 動物試驗與市售鮮乳之檢測-------------------------------51
第五章 討論--------------------------------------------------53
參考文獻------------------------------------------------------60

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