查詢結果分析
來源資料
頁籤選單縮合
題 名 | 利用單株與多株抗體開發歐索林酸酵素連結免疫吸附法檢驗試劑=Using Oxolinic Acid Polyclonal and Monoclonal Antibodies to Develop the Enzyme-linked Immunosorbent Assay Detection Kit |
---|---|
作 者 | 王渭賢; 陳柏叡; 林育興; 張鎮璿; 謝育錚; 涂青宇; 許添桓; 洪紹文; 洪紹文Shao-Wen Hung; | 書刊名 | 中國畜牧學會會誌 |
卷 期 | 45:1 2016.03[民105.03] |
頁 次 | 頁27-44 |
分類號 | 412.37 |
關鍵詞 | 抗體; 酵素連結免疫吸附法; 氟喹諾酮類; 歐索林酸; 殘留; Antibody; Enzyme-linked immunosorbent assay; Fluoroquinolone; Oxolinic acid; Residue; |
語 文 | 中文(Chinese) |
中文摘要 | 本研究目的在於開發直接競爭型酵素連結免疫吸附法之檢測試劑,期能精確、簡單及快速檢測禽畜水產品中歐索林酸( oxolinic acid, OA)殘留。結果顯示利用 N-hydroxysuccinimide ester 螯合法可成功製備 OA-human serum albumin(HSA)螯合物及藥物酵素結合體。另外,利用高效液相層析法搭配紫外光和螢光檢測器可成功地檢測 OA-HSA螯合物。運用所製備之 OA-HSA螯合物分別以皮下注射免疫紐西蘭白兔與腹腔注射免疫 BALB/c 小鼠各 6 次後,可分別成功地誘導產生血清中抗體力價高達 65,536 倍以上與 16,384 倍以上。之後,收集多株抗體( polyclonal antibody, pAb)與進行融合瘤實驗後獲得的單株抗體(monoclonal antibody, mAb),並分別製備成直接競爭型 ELISA檢測盤後,測得 OA 在磷酸緩衝液( phosphate buffer saline, PBS)中的最低檢測極限分別為 0.56 ppb(pAb)和 0.4 ppb(mAb)。OA pAb-ELISA檢測盤在牛奶、胎牛血清、魚肉、豬肉、雞肉、蝦肉及牛肉之 OA 最低檢測極限分別為 0.4、0.47、0.9、0.5、0.43、1.17 及 0.54 ppb;OA mAb-ELISA檢測盤在牛奶、 FBS、魚肉及豬肉之 OA 最低檢測極限分別為 0.37、0.43、0.86 及 0.51 ppb。OA pAb-ELISA檢測盤之組內(intra-assay)與組間(inter-assay)檢測變異值分別為 8.19% 和 6.70%。OA mAb-ELISA檢測盤之組內與組間檢測變異值分別為 7.46% 和 11.76%。OA 的融合瘤陽性率為 3.01%,檢測敏感度為 0.4 ppb。此外,交叉反應實驗的結果顯示, OA pAb-ELISA檢測盤對 amoxicillin 和 ampicillin 有極低的交叉反應率(小於 0.01%),OA mAb-ELISA檢測盤對所有檢測的抗生素亦均有極低的交叉反應率(小於 0.01%)。因此,綜合上述所有結果得知,本研究所開發之 OA-ELISA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 OA 之殘留檢驗。 |
英文摘要 | The aim of this study was to develop a simple, rapid, and reliable enzyme-linked immunosorbent assay (ELISA) for detecting the residues of oxolinic acid (OA) in edible animal tissues. By using the N-hydroxysuccinimide ester method, OA was conjugated with the carrier protein, human serum albumin (HSA). Furthermore, we successfully used HPLC with UV and fluorescence detectors to detect OA-HSA conjugates. In the production of OA polyclonal antibody (pAb) and monoclonal antibody (mAb), the antibody titer was 65,536 fold and 16,384 fold after subcutaneously or intraperitoneally injecting OA-HSA conjugates into New Zealand rabbits and BALB/c mice, respectively. After six boosters with OA-HSA conjugates, we collected antisera and cultural supernatant and detected their antibody sensitivites. The lowest detection limit (LDL) of OA pAb and mAb in PBS was 0.56 ppb and 0.4 ppb, respectively. Overwhelmingly, the OA ELISA kit so developed seemed to have a high sensitivity. In addition, the OA ELISA kit also had sufficient sensitivity for OA residue detection in different biological matrices. The coefficient variation values of the intra-assay and inter-assay of the OA pAb-ELISA kit and OA mAb-ELISA kit were 8.19% and 6.70% as well as 7.46% and 11.76%, respectively. In the tested matrixes of milk, FBS, fish meat, pork, chicken, shrimp, and beef, the LDLs of OA pAb were 0.4, 0.47, 0.9, 0.5, 0.43, 1.17 and 0.54 ppb, respectively. The LDLs of OA mAb were lower than 0.9 ppb in the tested matrixes. The 50% cross-reactivities of OA pAb and mAb for non-OA antibiotics were lower 0.01% or less, except for OA pAb cross-reacted with amoxicillin and ampicillin. Therefore, the OA ELISA kit so developed had a very good specificity. According to these results, the OA ELISA kit so developed was considered to be a simple, rapid, and reproducible tool for OA residue detection for the different animal productions. |
本系統中英文摘要資訊取自各篇刊載內容。