Development and Application the Rapid Detection Technique for the Residues of Fluoroquinolones

The aim of this project is to develop a rapid detection the fluoroquinolones in meats and feed with the immune reaction technique.The drug-protein combination, polyclone/or monoclone antibodies, immune screening, and hybridoma techniques will be used to reach our goal in this study.We try to use couple of proteins to bind with danofloxacin、enrofloxacin、ofloxacin and orbifloxacin to form the drug-protein compounds, respectively.Those compounds will be injected into rabbits or mice to produce polyclone and monoclone antibodies, respectively.The title and specify of antibodies are tested by the ELISA method.The serial dilution technique will be used for screening the identify of hybridoma.When all immune antibodies are dome, the test kit will be assembled and tested.一、 計畫目標: ( 1 )藥物蛋白質結合體的製備( 2 )免疫動物以獲得多株抗體或單株抗體融合瘤.( 3 )免疫篩選系統之建立.( 4 )細胞融合及篩選融合瘤細胞.二、 架構( 重要工作項目 ): ( 1 )藥物蛋白質結合體的製備: 依據Poni等( 1996 )、Bhargava等( 1999 )及Binodh等( 1999 )所述之不同藥物螯和蛋白質分別進行與danofloxacin、enrofloxacin、ofloxacin及orbifloxacin結合, 以進行動物免疫及免疫篩選系統之建立.( 2 )動物免疫以藥物衍生物蛋白質結合體分別免疫兔子進行多株抗體之製備或老鼠進行單株抗體融合瘤.( 3 )建立免疫篩選系統: 應用ELISA免疫篩選系統用來檢測所獲得之多株抗體力價及融合瘤之挑選.( 4 )細胞融合及篩選融合瘤細胞: 擬挑選對所有danofloxacin、enrofloxacin、ofloxacin及orbifloxacin藥物均具反應之融合瘤細胞.或對danofloxacin、enrofloxacin、ofloxacin及orbifloxacin等單一藥物具反應之融合瘤細胞.三、 預期效益: ( 1 )篩選出適合與藥物結合之蛋白質, 並建立製備技術.( 2 )獲得多株抗體或單株抗體融合瘤.( 3 )建立免疫篩選系統.( 4 )建立細胞融合及篩選融合瘤細胞技術

Development and Application the Rapid Detection Technique for the Residues of Fluoroquinolones-Polyclone and Monoclone Antibodies Production

1、B持鬮續穨改翵良}藥蘆物姣與P攜漹帶a蛋J白掑之抆螯g合X能鄐力O。C 依抩據渹Hammer等？(1995)、BDuan等？(2001)及哆Gabor等？(1995)所珥述z之岔藥蘆物恃螯g合X方閬式﹛，A繼~續穨改翵良}藥蘆物姣與P攜漹帶a蛋J白掑之尬熬鶡合X能鄐力O。C並癟利Q用咷Binodh等？(1999)之妞研膍究s，A期蟒能鄑利Q用峇不ㄕ同P攜漹帶a蛋J白敓改齔善衛藥蘆物咩抗凗體擗力O價糷及昵螯g合X效蘆果G。C2、B開}發o以Hliquid chromatography技瑋術N純瞻化？藥蘆物姣與P攜漹帶a蛋J白掑之抆螯g合X蛋J 白敓抗颩原鴗之壯技瑋術N。C 將N製s備？並籀透z析R完髡成角之岔藥蘆物垠衍l生耵物奕蛋J白桵質韏結畢合X體擖先？利Q用恲離髐子l交瘣換垓液G相蛩層h析R(ion exchange)進i行瘝粗吨分擢離鬫再A利Q用徆凝捐膠此過L濾o技瑋術N(gel filtration)進i一@步B純瞻化？螯g合X蛋J白捸，A以H期薛更饈能鉬誘今發o良}好n的漣抗傶藥蘆物咩抗凗體擗力O價貜的熔產ㄔ生矷。C(Bollag, et al.,1996)3、B抗颩原鴘注`射g免K疫怢兔艉子l工u作@。C 將N螯g合X並繪經gliguid chromatography純瞻化？之岔藥蘆物垠衍l生苀蛋J白桵質韏結畢合X體擃持鬮續穨免K疫怢兔艉子l以H進i行璁多h株閫抗凗體撉的獄製s備？。C(Hong, et al.,1989)4、B進i行瘜單畾株閫抗凗體暺製s備？。C 將N純瞻化？之岔藥蘆物垠衍l生耵物奕蛋J白桵質韏結畢合X體擏免K疫怞老揤鼠奎進i行瘜單畾株閫抗凗體橦融藻合X瘤F細茩胞M的瑪篩z選鵅，A擬嶼挑D選儮對鴭所狾有貫enrofloxacin、Bdanofloxacin、Bofloxacin及姛orbifloxacin藥蘆物咩均”具膉反狨應酗之尿融藻合X瘤F細茩胞M；F或庣對髾enrofloxacin、Bdanofloxacin、Bofloxacin及姛orbifloxacin等戊單璊一@藥蘆物咧具膉反狨應酗之尿融藻合X細茩胞M(Freshney, 2000)，A並簸參悁考涒Kitjaroentham等？(1998)、BFlens等？(1997)及呲Madersbacher等？(2000)所狶作@之妞研膍究s進i行瘜單畾株閫抗凗體撉的獄製s備？。C預w期螳效蠕益q：G1.建堨立葃藥蘆物姣與P攜漹帶a蛋J白淟螯g合X及巹純瞻化？技瑋術N2.提ㄟ高爬多h源膚抗凗體擗之坐力O價糷之壯技瑋術N3.建堨立葥高狗特S異妝性吨之妊單畾株閫抗凗體擗之壯技瑋術N。CThe aim of this project is to develop a rapid detection the fluoroquinolones in meats and feed with the immune reaction technique. The drug-protein conjugation, polyclonal or monoclonal antibodies, immune screening, and hybridoma techniques are studied in this study in this year. Successfully, we had produced antibodies from couple of proteins that bound with enrofloxacin, danofloxacin, ofloxacin, and orbifloxacin to form the drug-protein compounds in the last year, respectively. In this year we will try to enhance and increase the ratio of drug-protein conjugation. Those compounds will be purified by liquid chromatography technique. Those compounds will be injected into rabbits and mice to produce polyclonal and monoclonal antibodies, respectively. The title and specify of antibodies will be tested by the our set ELISA method. The titers of identified antibodies are going to be screened by the serial dilution technique

Oxolinic acid和Cipofloxacin藥物殘留檢驗試劑開發-多株及單株抗體之製備

Using our developed drug and carrier protein binding technique, the rapid residue detection kit for oxolinic acid and cipofloxacin will be studied. The poly- /and mono- antibodies will be produced from rabbits and hybrydoma cells. The test kits is going to set by using the above antiboies.利用本實驗室已開發之藥物與攜帶蛋白之螯合技術，針oxolinic acid和cipofloxacin二種藥物進行研究，應可得兔多價抗體及單株抗體。並且利用上述之抗體開發出檢測藥物殘留之快速檢測盤

Studies on the Mechanisms of the Anti-Bacteria Effects of Glycans in Fish in vivo

聚醣類為一種多磷脂醣類,存在於細菌細胞 壁、香茹、酵母菌及靈芝等等,其為一種非特 異性免疫促進劑,可以增進人類或動物體內之 免疫功能,其功效有抗細菌、抗病毒、抗黴菌 及抗腫瘤等等,而聚醣類之真正作用機制,至今 尚未十分清楚.本計畫擬使用魚類(如草魚、吳 郭魚等)為動物實驗模式,以不同之給予方式(如 口服及注射),評估目前常被使用及剛被萃取出 之不同聚醣類在魚體內之抗細菌效果,並且以 吞噬細胞指數、血清溶解酵素分析(Lysozyme assay )、補體活化分析及聚醣類受容器(Glucan receptor) 測定等試驗,來探討聚醣類在動物體內之真正 作用機制

Studies on the Primary Cell Culture from Invertebrates-Tiger Shrimp

本計畫依據原定之項目及進度正進行中,初 期之結果顯示:草蝦之幼苗(黑殼仔)細胞無法在 細胞培養基(MEM-20%FBS□Grace-20%FBS□TC-100-20%FBS及 TNM-FH-20%FBS)中存活,現正進行改變細胞培養基之 滲透壓研究,期能找出能適草蝦之幼苗細胞存 活之細胞培養基.由於健康草蝦幼苗之取得不 易,本計畫曾嘗試以泰國淡水長臂蝦之幼苗作 實驗,使用MEM-20%FBS□Grace-20%FBS□TC-100-20%FBS及 TNM-FH-20%FBS等細胞培養基,試建立初級細胞培養之方法及技術,結果顯示泰國淡水長臂蝦之幼 苗細胞可在Grace-20%FBS和MEM- 20%FBS細胞培養基中 成長分裂,甚至在MEM-20%FBS細胞培養基中可持續 分裂成長四次之久,但尚無法在細胞培養瓶中 長成單層細胞.該實驗正持續進展中,期在計畫 結束前有所新的結果

Effects of Antioxidative Vitamins and Minerals on Tilapia Macrophage

在魚類，吞噬細胞在對抗感染是非常重要的。有許多營養因子可影響到吞噬細胞的功能。在哺乳動物中，維生素C、維生素E 和一些抗氧化礦物質皆可調節免疫系統。但是在魚類，相關資料有關於維生素C、維生素E 和一些抗氧化礦物質在魚類吞噬細胞功能上之研究仍感不足。因此，我們非常感興趣在探討抗氧化維生素和抗氧化礦物質在魚類吞噬系統上所便演之角色

The Studies on the Monitoring and Controlling of a New Bacterial Disease of the Cultured Sturgeon

本研究計畫之目的在於監測和防治台灣養殖鱘魚的新型細菌性疾病。主要之工作項目：疾病之監控、病原之分離、細菌生物化學特性分析、PCR增幅該菌之16sDNA和16sDNA片段定序和親源性比對。完成本研究計畫後預期可了解此新型細菌性疾病在台灣養殖鱘魚之發生率，可藉由病原的生物化學和基因之特性鑑定分析可確診病原，同時所得知的成果可供未來對病原之致病機制和藥物防治方法之研究有極大之貢獻。The aims of this study are to monitor and control the new bacterial disease of cultured sturgeon in Taiwan. The main works include the new disease monitoring, bacterial isolation, bacterial biochemic characteristics identification, 16sDNA PCR amplification, and amplified 16sDNA segment sequencing and relationship mapping. The results will be very helpful for the bacterial pathogenicity and control by using antibacterial agent studies in the future