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標題: Fluoroquinolone類藥物酵素連結免疫吸附法殘留檢驗試劑之開發及應用
Development and Application of the Enzyme-Linked Immunosorbent Assay Residual Detection Kit for Fluoroquinolones
作者: 施清文
Shih, Ching-Wen
關鍵字: Fluoroquinolone;Fluoroquinolone;ELISA;Residual;酵素連結免疫吸附法;殘留
出版社: 獸醫學系
實驗結果顯示利用N-hydroxysuccinimide ester法可成功製備Eerofloxacin- BSA、Ofloxacin-BSA、Danofloxacin-BSA及Orbifloxacin- BSA藥物蛋白質衍生物結合體,並以相同的方法製備藥物-HAS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有效分析藥物-BSA結合體。試驗結果顯示,在抗體製備方面,經皮下免疫較脾內免疫更可有效誘發抗體力價的上升,經皮下免疫紐西蘭白兔3-4次後即可有效誘導抗體力價達32,768倍以上。
將收集的抗體製成直接競爭型ELISA盤後,在磷酸緩衝液(PBS),牛肉,牛奶,豬肉及魚肉中以danofloxacin檢測盤的檢測極限最低,分別為0.14, 0.6, 0.16, 0.61及0.62 ng/mL。在雞肉中則以orbifloxacin檢測盤的檢測極限最低,為1.33 ng/mL。在胎牛血清(FBS)中則以ofloxacin檢測盤的檢測極限最低,為0.27 ng/mL。danofloxacin、ofloxacin、orbifloxacin及enrofloxacin檢測盤在PBS檢測極限分別為0.14 , 1.18, 0.41, 2.38 ng/mL;在牛肉為0.60, 3.41, 0.64, 3.77 ng/mL;在雞肉為1.49, 1.33, 0.7, 3.61 ng/mL;在牛奶為0.16, 0.22, 0.21, 3.54 ng/mL;豬肉為0.61, 3.49, 0.66, 4.11 ng/mL;魚肉為0.62, 1.65, 0.78, 3.59 ng/mL;在FBS為1.18, 0.27, 0.3, 4.59 ng/mL。故所開發試劑的敏感度相當地高,未來可應用於臨床殘留之檢測。
由本研究結果顯示danofloxacin檢測盤之特異性最高,orbifloxacin檢測盤次之,ofloxacin檢測盤則再次之,以enrofloxacin檢測盤最低。在danofloxacin檢測盤中,其與enrofloxacin、norfloxacin、ofloxacin、orbifloxacin、oxolinic acid及sarafloxacin的交叉反應率均小於0.01%,在orbifloxacin檢測盤中,其與norfloxacin的交叉反應率為0.01%,而與danofloxacin、enrofloxacin、ofloxacin、oxolinic acid及sarafloxacin的交叉反應率均小於0.01%。在ofloxacin檢測盤中,其與enrofloxacin的交叉反應率為0.14%,而與danofloxacin、norfloxacin、orbifloxacin、oxolinic acid及sarafloxacin的交叉反應率均小於0.01%。在enrofloxacin檢測盤中,其與danofloxacin、norfloxacin、ofloxacin、orbifloxacin、oxolinic acid及sarafloxacin的交叉反應率分別為0.13%、0.18%、<0.01%、0.04%、<0.01%、0.02%。由以上結果可見藥物彼此間交叉反應率相當低,故本研究之檢測盤特異性很高。

The aim of this study is to develop a simple, rapid, and reliable enzyme-linked immunosorbent assay (ELISA) method for detecting the residues of the fluoroquinolones (enrofloxacin, ofloxacin, danofloxacin, and orbifloxacin) in the meats of fish, pork, beef, chicken, and milk.
By the N-hydroxysuccinimide ester method, the four tested chemical agents conjugated with BSA, HAS, and HRP had been done, respectively. Successfully, the tested conjugations were able to be detected by the HPLC with UV and fluoresce detector. Moreover, our results shown that the antibody titers by the subcutance immunization way was significantly higher than that intraspleen way in rabbit. Consequently, the titer of the subcuatance immunization was more than 32,768-fold after the fourth booster at four-weeks intervals.
The lowest detection limit (LDL) of the four fluoroquinolones (danofloxacin, ofloxacin, orbifloxacin, and enrofloxacin) in PBS, beef, chicken, milk, pork, fish, and FBS were (0.14, 0.60, 1.49, 0.16, 0.61, 0.62, 1.18 ng/mL), (1.18, 3.41, 1.33, 0.22, 3.49, 1.65, 0.27 ng/mL), (0.41, 0.64, 0.70, 0.21, 0.66, 0.78, 0.3 ng/mL), and (2.38, 3.77, 3.61, 3.54, 4.11, 3.59, 4.59 ng/mL) by the ELISA method, respectively. Therefore, the danofloxacin ELISA kit was the most sensitivity in our developed kits. However, it is sufficient sensitivity for the tested fluoroquinolones residues in biological matrices with these kits.
The rates of the cross-reaction of the developed danofloxacin, orbifloxacin, ofloxacin, and enrofloxacin ELISA kits to danofloxacin, enrofloxacin, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, and sarafloxacin were (100%, <0.01%, <0.01%, <0.01%, <0.01%, <0.01%, <0.01%), (<0.01%, <0.01%, 0.01%, <0.01%, 100%, <0.01%, <0.01%), (<0.01%, 0.14%, <0.01%, 100%, <0.01%, <0.01%, <0.01%), and (0.13%, 100%, 0.18%, <0.01%, 0.04%, <0.01%, 0.02%), respectively. Therefore, the above results shown that the best specificity of our developed kit was danofloxacin, then orbifloxacin, ofloxacin, and enrofloxacin. Especially, the antibody of danofloxacin did not have cross-reactivity for the other fluoroquinolones (enrofloxacin, norfloxacin, ofloxacin, orbifloxacin, and sarafloxacin). However, the other antibodies (enrofloxacin, ofloxacin, and orbifloxacin) had low cross-reaction with each other quionlones.
To elucidate the variability of standard curves for different assays, standard solutions were prepaerd independently with PBS and analyzed by the ELISA methods. The intra-assay and inter-assay coefficients of variation of danofloxacin, ofloxacin, enrofloxacin, and orbifloxacin ELISA kit were (3.35%, 3.51%), (2.93%, 4.35%), (8.16%, 10.1%), and (7.15%, 9.28%), respectively. Therefore, the best precision of our developed kit was danofloxacin, then ofloxacin, enrofloxacin, and orbifloxacin. From above assay results, our developed ELISA kits shown a simple, rapid, and reproducible method to detect the residues of the tested fluoroquinolones in the meats.
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