Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/13299
標題: Ciprofloxacin及oxolinic acid藥物酵素連結免疫吸附法殘留檢驗試劑之開發
Development of Enzyme-Linked Immunosorbent Assay Residual Detection Kit for Ciprofloxacin and oxolinic acid
作者: 陳柏叡
Chen, Bo-Rui
關鍵字: ELISA
酵素連結免疫吸附法
residue detection
polyclonal antibody
monoclonal antibody
殘留檢測
多株抗體
單株抗體
出版社: 獸醫學系暨研究所
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摘要: 本研究之目的在於嘗試以直接競爭型酵素連結免疫吸附法開發出一套簡單、快速、精確檢測禽畜產品中quinolone類抗菌劑ciprofloxacin (Cipro)及oxolinic acid (OA)的試劑。實驗結果顯示利用N-hydroxysuccinimide ester技術可以成功地製備Cipro-HSA及OA-HSA藥物蛋白質衍生物之結合體,並以相同的方法成功地製備藥物-BSA及藥物-HRP結合體。並成功地利用HPLC搭配紫外光及螢光檢測器可以偵測藥物-HSA結合體。由試驗結果顯示,經皮下免疫紐西蘭白兔六次後抗體力價可達65,536倍以上,而老鼠經六次免疫即誘導產生抗體力價高達16,384倍以上。將多株及單株抗體收集製備成直接競爭型ELISA盤後,以Cipro檢測盤(多株及單株)在磷酸緩衝液PBS的檢測極限為0.78 ng/mL和0.82 ng/mL;而在牛奶、胎牛血清 (FBS)、魚肉、豬肉、雞肉、蝦肉及牛肉等基質中其檢測極限分別為1.08、1.37、0.83、1.1、0.88、1.32及1.14 ppb,單株抗體檢測盤其檢測敏感度皆可低於1.03 ppb。而OA檢測盤(多株及單株)在磷酸緩衝液PBS為0.56 ng/mL和0.4 ppb,在牛奶、FBS、魚肉、豬肉、雞肉、蝦肉和牛肉等基質的檢測極限分別為0.4、0.47、0.9、0.5、0.43、1.17及0.54 ppb,單株抗體檢測盤其檢測敏感度皆可低於0.9 ppb。 Cipro檢測盤之組內 (intra-assay) 與組間 (inter-assay) 檢測變異值分別為5.82%和7.38%;而OA檢測盤次之其組內與組間檢測變異值分別為8.19%及6.70%。單株抗體 (mAb)檢測盤的製備結果顯示,抗Cipro及抗OA的融合瘤陽性率分別為4.16%和3.01%,檢測敏感度為為0.82 ppb和0.4 ppb。mAb Cipro檢測盤其組內與組間變異係數值為9.58%和7.38%,而mAb OA檢測盤組內與組間之變異係數值為7.46%和11.76%。交叉反應試驗結果顯示,除了Cipro檢測盤對enrofloxacin (58%)及norfloxacin (37%)有較高的反應率外,其餘皆小於1。組內結果顯示,所製備的mAb檢測盤對其他藥物之交叉反應率皆低於0.005。因此,綜合上述所有結果顯示,本研究所開發之檢測試劑具有簡單、快速及精確之特性,可應用於現場針對禽畜水產品Cipro及OA類抗菌劑之殘留檢驗。
The aim of this study is to develop a simple, rapid, and reliable enzyme-linked immunosorbent assay (ELISA) method for detecting the residues of ciprofloxacin (Cipro)and oxolinic acid (OA)in the varietal samples. By using the N-hydroxysuccinimide ester method, the Cipro and OA was productively conjugated with HSA, BSA, and HRP, respectively. Successfully, the tested conjugations were able to be detected by the HPLC with UV and fluoresce detectors. The study showed that the titers of the serum antibodies from rabbit and mice were 65,536-fold and 16,384-fold after six boosters, respectively. The lowest detection limit (LDL) of the Cipro and OA(polyclone and monoclone antibody) in PBS was 0.78 ng/mL and 0.56 ng/mL by the ELISA method, respectively. The coefficient variation value of the intra-assay and inter-assay of the polyclonal and monoclonal antibodies (mAb) Cipro ELISA kit were (5.82%, 7.38%) and (9.58%, 7.46%), respectively. According our results, the LDL of the Cipro ELISA and OA ELISA kits in milk, Fetal Bovine Serium, fish, pork, chicken, shrimp, and beef meats were (1.08 ppb, 0.4 ppb) , (1.37 ppb and 0.47 ppb), (0.83 ppb and 0.9 ppb), (1.1 ppb and 0.5 ppb), (0.88 ppb and 0.43 ppb), (1.32 ppb and 1.17 ppb), and (1.14 ppb and 0.54 ppb), respectively. Meanwhile, our data shown that the cross reaction ratioes of the both kits to frequently used chemical agents were below 1%, except the Cipro ELISA Kits to enrofloxacin (58%) and norfloxacin (37%). From the mAb tests, our results indicated that the positive hybridoma ratio for anti-Cipro and anti-OA was 4.16% and 3.01%, respectively. The coefficient variation values of the intra-assay and inter-assay of OA ELISA kit and mAb OA ELISA kit were (8.19%, 6.706%) and (8.20%, 11.76%), respectively. Our result indicate the LDL of the mAb-Cipro ELISA and mAb-OA ELISA kits in milk, Fetal Bovine Serium, fish, and pork were (0.93 ppb, 0.37 ppb) , (1.03 ppb and 0.43 ppb), (0.87 ppb and 0.86 ppb), (0.85 ppb and 0.51 ppb), respectively. We found the cross-reactive ratio of the OA ELISA plate to our tested non-quinolones was below 0.01%. Our results suggested the LDL of the mAb Cipro and mAb OA in PBS were 0.82 ppb and 0.4 ppb. Meanwhile, the cross-reacted ratio of the bothe mAb Cipro and mAb OA ELISA plates to our tested agents was 0.005. Therefore, our developed Cipro and OA ELISA kit had very good precision. According to these results, our developed Cipro ELISA kit seemed to be a simple and reproducible tool for Cipro residue detection for the different animal productions.
URI: http://hdl.handle.net/11455/13299
其他識別: U0005-2812200615190700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2812200615190700
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