Ciprofloxacin藥物酵素連結免疫吸附法殘留檢驗試劑之開發

Abstract

本研究目的在於開發直接競爭型酵素連結免疫吸附法，期能精確、簡單及快速檢測禽畜水產品中ciprofloxacin (Cipro)殘留之檢測試劑。實驗結果顯示利用N-hydroxysuccinimide ester法可成功製備Cipro-human serum albumin (HSA)之結合蛋白，同時以相同的方法可以分別成功製備出藥物-bovine serum albumin結合蛋白和藥物-horseradish peroxidase之結合體。另外，利用高效液相層析法(high performance liquid chromatography)搭配紫外光和螢光檢測器可以檢測藥物-HSA結合體。運用所製備之Cipro-HSA之結合蛋白以皮下免疫紐西蘭白兔六次後，可成功誘導產生多株抗體，力價高達65,536倍以上。收集多株抗體並製備成直接競爭型ELISA盤後，在磷酸緩衝液 (PBS)中的最低檢測極限為0.78 ppb，而在牛奶、fetal bovine serum、吳郭魚肉、猪肉、雞肉、蝦肉及牛肉中分別為1.08 ppb、1.37 ppb、0.83 ppb、1.1 ppb、0.88 ppb、1.32 ppb 及1.14 ppb，故所開發試劑的敏感度相當高，未來應可用於臨床Cipro殘留之檢測。在精確度方面，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％，顯示Cipro檢測盤精確度高。此外，本檢測盤對enrofloxacin和norfloxacin有較高的反應率外，對其餘抗菌劑皆小於0.2％。

The aim of this study is to develop a simple, rapid, and reliable enzyme-linked immunosorbent assay (ELISA) for detecting the residues of ciprofloxacin in the edible animal tissues. By the N-hydroxysuccinimide ester method, ciprofloxacin was conjugated with carrier proteins, HSA and HRP, respectively. Furthermore, we successfully used HPLC with UV and fluorescence detectors to detect ciprofloxacin-HSA conjugated protein. In the production of ciprofloxacin polyclonal antibody, the antibody titer was reached over 65,536 fold after subcutaneously injected ciprofloxacin-HSA conjugated protein into New Zealand rabbits. After six boosters with ciprofloxacin-HSA conjugated protein, we collected antisera and detected antibody sensitivity. The lowest detection limit (LDL) in PBS was 0.78 ppb. Overwhelmingly, our developed ciprofloxacin ELISA kit seemed to have very high sensitivity. At the same time, ciprofloxacin ELISA kit appeared to their sufficient sensitivity for ciprofloxacin residue detection in differnet biological matrices. The coefficient variation values of the intra-assay and inter-assay of the ciprofloxacin ELISA kit was 5.82% and 7.38%. In the other matrix (milk, FBS, fish, pork, chicken, shrimp, and beef), the LDL was 1.08, 1.37, 0.83, 1.1, 0.88, 1.32, and 1.14 ppb, respectively. In the 50% cross-reactivety for non-Cipro antibiotics was below 0.2%, except the to enrofloxacin and norfloxacin. Therefore, our developed ciprofloxacin ELISA kit had very good precision. According to these results, our developed ciprofloxacin ELISA kit seemed to be a simple, rapid, and reproducible tool for ciprofloxacin residue detection for the different animal productions.