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研究生:邱慶豐
研究生(外文):Ching-Feng Chiu
論文名稱:霍亂毒素多株及單株抗體之生產與其免疫分析試片之開發研究
論文名稱(外文):Studies on the Development of Production of Polyclonal and Monoclonal Antibodies against Cholera Toxin and Immunoassay Strip for Cholera Toxin
指導教授:周淑芬周淑芬引用關係
指導教授(外文):Shu-Fen Chou
學位類別:碩士
校院名稱:嘉南藥理科技大學
系所名稱:生物科技系暨研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:114
中文關鍵詞:免疫分析試片細胞融合單株抗體多株抗體霍亂毒素免疫層析試紙
外文關鍵詞:immunochromatographic stripcholera toxinpolyclonal antibodiesmonoclonal antibodiesimmunoassay stripcell fusion
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本研究目的以霍亂毒素(cholera toxin, CT)免疫小鼠來生產及純化霍亂毒素多株與單株抗體,應用霍亂毒素多株抗體建立霍亂毒素酵素連結免疫分析法(enzyme-linked immunosorbent assay, ELISA),並開發霍亂毒素單株抗體於免疫分析試片(immunoassay strip)檢測上。傳統檢測霍亂毒素以逆相被動乳膠凝集法(reverse passive latex agglutination, RPLA)檢測毒素蛋白質和利用聚合酶鏈鎖反應法(polymerase chain reaction, PCR)檢測毒素基因為主。RPLA雖能檢測出1-2 ng/mL CT,但易受檢體中其他腸內毒素而影響其準確性,且需費時20-24小時才能判讀結果。而PCR檢測霍亂毒素基因雖較免疫分析法靈敏且快速,但易造成誤判不具分泌霍亂毒素能力之非產毒型霍亂弧菌。
本研究首先進行霍亂毒素多株及單株抗體之生產與純化。小鼠腹水抗體使用Hitrap™ rProtein A Column純化出霍亂毒素多株抗體,經間接型ELISA免疫分析可有效檢測CT濃度為0.05 µg/mL,且具低交叉反應性。實驗成 篩選出五株高效價之單株抗體細胞株,分別為CT-3A、CT-4C、CT-11D、CT-11H及CT-9A,經鑑定其單株抗體同種型(isotyping)均屬於IgM重鏈,λ輕鏈。其中CT-3A與CT-9A單株抗體經大量生產以Hitrap™ IgM Purification Column純化後,經間接型ELISA免疫分析抗體效價在1:15,625。第二部分進行膠體金(colloidal gold)合成工作,並成 將霍亂毒素單株抗體與20 nm膠體金進行抗體染色,進一步應用於霍亂毒素免疫分析試片之開發上。實驗結果此霍亂毒素免疫分析試片在40-100 µg/mL CT間呈一線性關係且無交叉反應出現,並可應用於實際樣品檢測上,檢測所需時間在15-20分鐘。
The objective of this study is to develop an immunoassay strip (immunochromatographic strip) for cholera toxin (CT) using polyclonal and monoclonal antibodies against CT produced. In this study, the enzyme-linked immunosorbent assay (ELISA) for CT was set up using anti-cholera toxin polyclonal antibodies (anti-CT pAb), and the CT immunoassay strip using anti-cholera toxin monoclonal antibodies (anti-CT mAb) was developed. Conventional methods for determining this CT is to use reverse passive latex agglutination (RPLA) to determine CT protein and use polymerase chain reaction (PCR) to determine CT gene. Although RPLA can determine 1-2 ng/mL of CT protein, it can effect its accuracy by other enterotoxin of samples and took 20~24 hours. PCR has more sensitive and rapid than other immunoassays, but it is easy to mistake the non-toxin gene in other V. cholerae.
Firstly, this study was to produce and purify polyclonal and monoclonal antibodies against CT. The experiment result was that mouse anti-CT pAb from mouse ascites purified by Hitrap™ rProtein A Column. This anti-CT pAb could determine 0.05 µg/mL of CT and low cross-reaction. The five high-titer anti-CT mAb-producing hybridoma cell lines selected and designated CT-3A, CT-4C, CT-11D, CT-11H, and CT-9A. The isotypes of the five anti-CT mAbs were identified as IgM heavy chain and λ light chain. For the purification of anti-CT mAb, Hitrap™ IgM Purification Column was used. The highest titer of anti-CT mAb (CT-3A and CT-9A) determined by indirect ELISA was 1:15,625. Secondly, we made the colloidal gold from the chloroauric acid (HAuCl4) and labeled anti-CT mAb with the 20 nm colloidal gold. This anti-CT mAb immunoassay strip set up could determine in 40-100 µg/mL CT and without cross-reaction. Moreover, this strip could be applied to test samples in fact, and detection time was approximately 15-20 minutes.
中文摘要 ………………………………………………………………Ⅰ
英文摘要 ………………………………………………………………Ⅱ
致謝 ……………………………………………………………………Ⅲ
目錄 ……………………………………………………………………Ⅳ
圖表目錄 ………………………………………………………………Ⅶ


第一章、前言 …………………………………………………………1
1-1、研究動機…………………………………………………………1
1-2、霍亂弧菌…………………………………………………………3
1-3、霍亂毒素…………………………………………………………5
1-4、免疫分析原理……………………………………………………14
1-5、多株及單株抗體之原理及應用…………………………………20
1-6、免疫分析試片……………………………………………………27

第二章、材料與方法 …………………………………………………34
2-1、材料………………………………………………………………34
2-1-1、實驗動物、骨髓癌細胞株與三株腸內桿菌…………………34
2-1-2、試劑……………………………………………………………34
2-1-3、器材與裝置……………………………………………………36
2-2、免疫分析法………………………………………………………37
2-2-1、酵素連結免疫分析法篩選系統之建立………………………37
2-2-2、免疫墨點分析法之建立………………………………………38
2-3、霍亂毒素多株抗體製備…………………………………………39
2-3-1、ICR小鼠免疫計畫 ……………………………………………39
2-3-2、血清抗體檢測…………………………………………………40
2-3-3、腹水抗體純化…………………………………………………41
2-3-4、腸內桿菌培養…………………………………………………42
2-4、霍亂毒素單株抗體製備…………………………………………43
2-4-1、BALB/c小鼠免疫計畫…………………………………………43
2-4-2、血清抗體檢測…………………………………………………43
2-4-3、NS-1骨髓癌細胞培養…………………………………………46
2-4-4、細胞融合………………………………………………………47
2-4-5、單株抗體同種型 (isotyping)測定…………………………52
2-4-6、單株抗體大量生產……………………………………………52
2-4-7、單株抗體純化…………………………………………………53
2-5、免疫分析試片製作………………………………………………55
2-5-1、膠體金合成……………………………………………………55
2-5-2、膠體金粒子與抗體結合最適活性之測定……………………55
2-5-3、膠體金抗體反應與純化………………………………………56
2-5-4、試片製作………………………………………………………57

第三章、結果與討論 …………………………………………………58
3-1、霍亂毒素多株抗體………………………………………………58
3-1-1、霍亂毒素多株抗體之生產與純化……………………………58
3-1-2、霍亂毒素多株抗體效價曲線與霍亂毒素檢量線……………59
3-1-3、腸內桿菌交叉反應之測定……………………………………60
3-1-4、模擬菌液檢體之霍亂毒素檢測………………………………61
3-2、霍亂毒素單株抗體………………………………………………62
3-2-1、BALB/c小鼠免疫………………………………………………62
3-2-2、細胞融合………………………………………………………62
3-2-3、融合瘤細胞篩選與單株化效價測定…………………………63
3-2-4、霍亂毒素單株抗體生產與純化………………………………66
3-3、免疫分析試片……………………………………………………68
3-3-1、膠體金合成……………………………………………………68
3-3-2、單株抗體與膠體金吸附反應…………………………………69
3-3-3、分析試片製作條件……………………………………………70
3-3-4、霍亂毒素免疫分析試片之檢量線……………………………71
3-3-5、分析試片交叉反應……………………………………………72
3-3-6、實際樣品之檢測………………………………………………73

第四章、結論…………………………………………………………100

參考文獻………………………………………………………………102


圖表目錄

表1-1 不同霍亂弧菌的血清型、亞型、遺傳型和產生霍亂毒素之比較 ……………………………………………………………………5
表1-2 AB5腸毒素家族蛋白之差異比較 …………………………13
表1-3 人類免疫球蛋白之物理化學性質…………………………18
表1-4 膠體金粒徑大小與其外觀顏色之特性比較………………31
表2-1 單株抗體生產之時間表……………………………………45
表2-2 抗體與膠體金之最適反應測試……………………………57
表3-1 霍亂毒素濃度、顯色峰度及像素強度之統計表…………72
表3-2 霍亂毒素免疫分析試片之實際樣品檢測…………………73


圖1-1 霍亂毒素(CT)和熱不穩定型腸毒素(LT)之蛋白質立體結構 ……………………………………………………………………6
圖1-2 霍亂毒素之B次單元蛋白質結構圖 ………………………7
圖1-3(A) 霍亂毒素與小腸黏膜細胞外膜上GM1受器之作用圖…9
圖1-3(B) 霍亂毒素影響腸黏膜細胞膜上G蛋白之分子作用圖…10
圖1-3(C) 霍亂毒素進入腸黏膜細胞後活化Gsa-GTP複合體 ……11
圖1-4 人類免疫球蛋白之基本單元結構…………………………16
圖1-5 抗原抗體免疫檢測分析類別………………………………19
圖1-6 多株抗體製備過程…………………………………………21
圖1-7 單株抗體製備過程…………………………………………22
圖1-8 單株與多株抗體生產及其特異性之比較…………………24
圖1-9 HAT培養液選殖原理 ………………………………………25
圖1-10 免疫層析試紙之構造圖 …………………………………29
圖1-11 免疫層析試紙檢測示意圖 ………………………………30
圖1-12 膠體金與抗體的鍵結方式 ………………………………33
圖2-1 小鼠腹腔注射………………………………………………40
圖2-2 小鼠尾靜脈採血……………………………………………41
圖2-3 BALB/c小鼠免疫計畫………………………………………46
圖2-4 小鼠解剖圖…………………………………………………49
圖2-5 融合瘤細胞擴大培養圖……………………………………52
圖3-1 腹腔免疫霍亂毒素之ICR小鼠血清效價檢測圖 …………74
圖3-2 以HitrapTM rProtein A親和性管柱純化霍亂毒素ICR小鼠腹水之親和層析圖 ……………………………………………………75
圖3-3 霍亂毒素多株抗體(anti-CT pAb)純化完成後之抗體效價曲線 ……………………………………………………………………76
圖3-4 霍亂毒素多株抗體(1:1,000)對不同濃度霍亂毒素之檢量線 ……………………………………………………………………77
圖3-5 霍亂毒素多株抗體(1:1,000)與EHEC、ETEC和S.D.1培養菌液之交叉反應圖 ……………………………………………………78
圖3-6 APW培養液含不同濃度霍亂毒素之免疫墨點檢測 ………79
圖3-7 APW培養液含不同濃度霍亂毒素之ELISA檢測……………80
圖3-8 腹腔免疫霍亂毒素之BALB/c小鼠血清效價檢測圖………81
圖3-9 NS-1骨髓癌細胞與脾臟細胞之生長情形…………………82
圖3-10 NS-1骨髓癌細胞與脾細胞融合瘤細胞生長照片 ………83
圖3-11 分泌霍亂毒素單株抗體之融合瘤細胞單株化後第七天(D7)及第十天(D10)細胞生長照片……………………………………84
圖3-12 分泌高效價霍亂毒素單株抗體融合瘤細胞株之篩選 …85
圖3-13 小鼠融合瘤同種型鑑定試紙鑑定霍亂毒素單株抗體類型.86
圖3-14 以HitrapTM IgM Purification Column親和性管柱純化霍亂毒素單株抗體之BALB/c小鼠腹水之親和層析圖 …………………87
圖3-15 CT-3A及CT-9A純化完成之anti-CT mAb抗體效價曲線…88
圖3-16 不同粒徑膠體金之外觀顏色差異及試片上顯色差異……89
圖3-17(A) 膠體金粒徑測定分析圖………………………………90
圖3-17(B) 膠體金粒徑測定分析圖………………………………91
圖3-17(C) 膠體金粒徑測定分析圖………………………………92
圖3-18 膠體金與抗體反應後外觀顏色變化及游離之抗體測定.93
圖3-19 免疫分析試片之抗體固定條件測試 ……………………94
圖3-20 霍亂毒素免疫分析試片檢測結果 ………………………95
圖3-21 霍亂毒素免疫分析試片顯色區塊分析結果 ……………96
圖3-22 霍亂毒素免疫分析試片之檢量線 ………………………97
圖 3-23(A) 霍亂毒素免疫分析試片之牛血清白蛋白、C反應蛋白(CRP)與黃麴毒素交叉反應測試…………………………………98
圖3-23(B) 霍亂毒素免疫分析試片之人類血清與三株腸內菌(ETEC, EHEC, and S.D.)交叉反應測試 ………………………99
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