人類干擾素-γ抗體之生產及雙弁鄑K疫層析試片系統之開發

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Title:	人類干擾素-γ抗體之生產及雙弁鄑K疫層析試片系統之開發 Development of Human Interferon-γ Antibodies Production and Bifunctional Immunochromatographic Strip
Authors:	余欣嶸 Hsin-Jung Yu
Contributors:	周淑芬嘉南藥理科技大學：生物科技研究所
Keywords:	干擾素-γ Interferon-γ
Date:	2006
Issue Date:	2008-11-24 17:01:36 (UTC+8)
Abstract:	干擾素 ( Interferon, IFN ) 屬於細胞激素的成員之一，具重要免疫調節弁遄A會因病毒感染細胞而被誘發生成，並抑制人體內病毒的複製與細胞生長，是抵抗多種病毒感染的第一道防線。干擾素-γ ( Interferon-γ, IFN-γ ) 經研究證實除抗病毒能力外也具有抑制腫瘤的弁遄A其免疫調節能力比其他干擾素為佳，也是重要的醫療蛋白之一。本研究的目的主要是人類IFN-γ多株與單株抗體之生產，並建立人類IFN-γ抗體之鑑定分析；同時應用其IFN-γ及IFN-ㄖ凗撽鬋虪能免疫層析試片( bifunctional immunochromatographic strip ) 之開發上。本研究所開發之試片可應用於居家照護 ( home care ) 或護理站 ( point-of-care ) 之實際樣品檢測，極具應用價值。本研究首先進行 IFN-γ 多株及單株抗體之生產與純化。實驗成正z選出11株高效價之單株抗體融合瘤細胞株，分別為 Iγ-A8、Iγ-A10、Iγ-B9、Iγ-C6、Iγ-E3、Iγ-F8、Iγ-F9、Iγ-G4、Iγ-G6、Iγ-H6 及 Iγ-H9，經鑑定其單株抗體同種型 ( isotype ) 全屬於IgG2a 重鏈及 κ 輕鏈。其中 Iγ-F8 單株抗體經大量生產後，以 Hitrap™ rProtein A Fast Flow Column 純化，經間接型 ELISA 分析抗體效價為1：15,625。純化後之Iγ-F8經由SDS-PAGE鑑定分析顯示其為高純度之單株抗體，並具有人類IFN-γ 專一性辨識能力，因此本研究所生產之IFN-γ單株抗體具有特異性高、純度高、效價高等優點。第二部分進行膠體金粒子 ( colloidal gold particles ) 之合成工作，並成弗NIFN-?及 IFN-γ 單株抗體與25 nm膠體金結合，成扒}發出雙弁鄑K疫層析試片。實驗結果顯示此雙弁鄑K疫層析試片能分別於緩衝溶液及小鼠血清中檢測人類 IFN-ㄓ峇H類IFN-γ，且無交叉反應出現，檢測所需時間約5分鐘。本研究所製備之可拋棄式免疫層析試片，具有以下優點：1.樣品不需前處理；2.所需樣品量少；3.可同時檢測二種標的物；4.操作方便；5.檢測快速；6.低成本；7.有效檢測；8.可長期保存；9.不需要任何儀器之使用。因此本研究所製備之可拋棄式免疫層析試片極具應用價值。 Interferons ( IFNs ) play a role in inhibition of tumor growth and participate in immunoreactions. Among IFNs, interferon-γ ( IFN-γ ) is one of the most important therapeutic protein and its immunodulation ability is better than that of other types. In this study, mice were immuned by injecting with human IFN-γ, then the polyclonal and monoclonal antibodies against human IFN-γ were produced and detection system of monoclonal antibodies against human IFN-γ ( anti-IFN-γ mAbs ) was established. The bifunctional immunochromatographic strip using anti-IFN-γ and anti-IFN-?antibodies was also developed. This self-assembled immunochromatographic strip will be used for home care and point-of-care in clinical diagnosis. Firstly, this study was to produce and purify anti-IFN-γ pAbs and anti-IFN-γ mAbs. Eleven high-titer anti-IFN-γ mAb-producing hybridoma cell lines were obtained and designated Iγ-A8, Iγ-A10, Iγ-B9, Iγ-C6, Iγ-E3, Iγ-F8, Iγ-F9, Iγ-G4, Iγ-G6, Iγ-H6 and Iγ-H9. These isotypes of mAbs were identified as IgG2a heavy chain and κ light chain. For the purification of anti-IFN-γ mAbs, Hitrap™ rProtein A Fast Flow column was also used. The highest titer of anti-IFN-γ mAb ( Iγ-F8 ) determined by indirect ELISA was 1:15,625. The purity analysis of the anti-IFN-γ mAbs was determined using SDS-PAGE. The results demonstrated the purified Iγ-F8 obtained were of high purity. The anti-IFN-γ mAbs could specifically recognize the IFN-γ. Secondly, the colloidal gold was made from the chloroauric acid ( HAuCl4 ) and labeled anti-IFN-?and anti-IFN-γ mAbs with 25 nm of colloidal gold. This bifunctional immunochromatographic strip could simultaneously determine two marker：human IFN-γ and IFN-? and had no cross-reaction. The detection time was approximately 5 minutes. The advan tages of self-assembled immunochromatographic strip was no pretreatment, low sample requirement, bifunction, easy operation, rapid determination, low price, no cross-reaction, long-term preservation, no machine needed etc.
Relation:	校內一年後公開，校外永不公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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