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研究生: 詹豐碩
Feng-So Chan
論文名稱: 對應人類血紅素的IgY抗體生產、純化與分析研究
Production, purification and characterization of chicken IgY directed to human hemoglobin
指導教授: 王玉麒
Wang, Yu-Chie
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2014
畢業學年度: 102
語文別: 中文
論文頁數: 151
中文關鍵詞: 大腸癌人類血紅素免疫法糞便潛血檢查吸附管柱IgY
英文關鍵詞: Colorectal cancer, human hemoglobin, immunochemical fecal occult blood test, affinity column, IgY
論文種類: 學術論文
相關次數: 點閱:181下載:1
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  • 「大腸癌」為當前世界各國最盛行的癌症之一。定期的糞便潛血檢查可及早發現大腸癌徵兆,因而有助於降低其發生率與死亡率。本研究之目的即針對免疫糞便潛血檢驗試劑中的核心抗體,進行以雞隻為實驗動物的生產平台測試。
    在測試內容上,我們一方面評估抗原劑量、佐劑、及激活注射對雞隻體液免疫反應的影響,另一方面也監測血清及蛋黃中抗體效價的變化情形,進而歸納出抗體生產的適當條件。在抗體純化部分,我們除了製備抗原管柱之外,也發展出效化清理抗原管柱的步驟;以該管柱吸附純化的專一性抗體,無論純度與活性均明顯優於市售抗體,並且不會辨識血紅素以外的蛋白質。此外,我們也對抗體應用的敏感度及是否受食源血紅素及上消化道出血的干擾進行初期評估。總體而言,以雞隻生產對應人類血紅素的抗體,不僅花費成本較低廉、操作較為方便、產量較高、過程更較符合動物福祉法規,所生產的IgY抗體也有良好的作用專一性與敏感度,因此具有取代當前業界使用的兔產抗體的潛力。

    Colorectal cancer (CRC) is one of the most prevalent cancers in the world. Routine fecal occult blood tests (FOBT) help detect CRC in its early stages and thus decrease CRC mortality and incidence. The aim of this study is to establish a production platform, using chickens as experimental animals, for the core component of anti-human hemoglobin antibody (anti-hHb Ab) used in the immunochemical FOBT (I-FOBT). To that end, we have examined the effects of antigen dose, adjuvant and booster injection on chicken’s humoral immune response and monitored changes of antibody titers in the sera and yolksfrom that the optimal parameters for anti-hHb Ab production can be deduced. As for antibody purification, we have prepared affinity column with antigen-coupled beads and developed an effective cleaning procedure for that column. The affinity-purified sample of anti-hHb IgY does not cross-react with non-hemoglobin proteins and exhibits superior purity and reactivity to antigen than that of commercially available. In addition, preliminary assessments have been performed to evaluate the sensitivity of using anti-hHb IgY in a biochemical assay and to scrutinize the plausible interferences exerted by animal blood-containing food or by upper gastrointestinal tract bleeding. In conclusion, the use of chickens for anti-hHb Ab production brings many advantages over the conventional mammal hosts, including lower cost, higher yield, easier handling process and greater compliance to animal welfare regulations. Since the genarated anti-hHb IgY reveals great sensitivity and specificity in biochemical assays, they can be considered as an excellent alternative for the rabbit anti-hHb IgG currently used in the commercial I-FOBT kits.

    摘要 1 Abstract 2 壹、緒論 4 一、 大腸癌對民眾健康的威脅 4 二、 大腸癌篩檢的重要性 6 三、 大腸癌的篩檢方式 8 四、 糞便潛血檢查 11 五、 雞隻抗體多樣性的機制 18 六、 蛋黃抗體的來源 22 七、 IgY的分子結構與特性 24 八、 雞隻蛋黃IgY抗體的生產與應用 27 九、 研究目的 34 貳、材料與方法 35 一、 人類血紅素抗原樣品的配製與分析 35 二、 其他血紅素樣品的製備 35 三、 非血紅素蛋白的製備 36 四、 人類血紅素的水解分析 36 五、 蛋白質定量(Bradford assay) 37 六、 雞隻的抗原免疫注射 37 七、 雞隻的靜脈抽血 38 八、 SDS膠體電泳(SDS-PAGE)分析 39 九、 免疫轉漬分析(Immunoblotting; Western blotting) 39 十、 酵素連結免疫吸附法分析(enzyme-linked immunosorbent assay; ELISA) 40 十一、 抗原吸附管柱的製備與清理處置 41 十二、 Chicken anti-hHb 抗體的萃取與純化 45 十三、 統計與分析 46 參、結果 47 一、 對應人類血紅素(hHb)的IgY生產 47 (1) hHb抗原的純度與光學特性分析 47 (2) 雞隻的免疫注射及採樣 51 (3) 雞隻的血清效價變化情形 52 (4) 雞隻的蛋黃效價變化情形 56 (5) 實驗雞隻的產蛋率變化情形 61 二、 Chicken anti-hHb IgY的萃取與純化 63 (1) 蛋黃IgY抗體的萃取 63 (2) 一號抗原管柱的製備與使用 63 (3) 抗原管柱的清理測試 71 (4) 二號抗原管柱的製備與使用 72 三、 Anti-hHb IgY的測試結果 79 (1) 敏感度分析 79 (2) 專一性分析 81 (3) 高溫及蛋白酶處理對hHb抗原性(antigenecity)的影響 87 (4) 與外購抗體的比較 93 肆、討論 97 一、 Anti-hHb IgY抗體的生產 97 (1) 雞隻的免疫注射條件 97 (2) 血清與蛋黃抗體效價的變化情形 102 (3) 蛋黃抗體效價的影響因素探討 103 (4) Anti-hHb抗體的生產條件建議 107 二、 Anti-hHb IgY抗體的萃取與純化 109 (1) 應用抗原管柱的問題發現 111 (2) 應用抗原管柱的問題解決 114 (3) 專一性抗體比例的探討 115 三、 Anti-hHb IgY的測試 120 (1) 敏感度 120 (2) 專一性 120 (3) 生化處理後的hHb抗原性變化 122 伍、總結 124 參考文獻 125 附錄 143 附錄一、縮寫對照表 143 附錄二、中英對照表 145 附錄三、常用溶液配方 148 圖目錄 圖一、G-FOBT糞便潛血檢測的示意圖 12 圖二、I-FOBT糞便潛血檢測的示意圖 14 圖三、基因轉換的可能機制 20 圖四、雞隻抗體基因的基因重組與基因轉換示意圖 21 圖五、雞蛋在發育期間,不同類型抗體的轉移與差異性分布情形 23 圖六、IgY與IgG的結構比較圖 25 圖七、抗原管柱的清理處置預試驗示意圖 42 圖八、抗原管柱的清理處置示意圖 44 圖九、人類血紅素樣品的純度分析 48 圖十、人類血紅素樣品的光譜分析 50 圖十一、雞隻的免疫注射期程及血清與蛋黃樣品的收集時間點 51 圖十二、雞隻血清的抗hHb效價分析結果 54 圖十三、雞隻蛋黃的抗hHb效價分析結果 58 圖十四、雞隻血清與蛋黃的抗hHb效價的相對變化情形 60 圖十五、四組雞隻於實驗期間的週產蛋率變化情形 62 圖十六、一號抗原管柱的抗體純化結果 65 圖十七、一號抗原管柱純化前、後的抗體樣品活性比較 67 圖十八、一號抗原管柱所純化anti-hHb IgY樣品的分析 70 圖十九、二號抗原管柱的抗體純化結果 74 圖二十、二號抗原管柱純化前、後的抗體樣品活性比較 76 圖二十一、二號抗原管柱所純化抗體樣品的光譜分析 78 圖二十二、Anti-hHb IgY的敏感度分析結果 79 圖二十三、Anti-hHb抗體對肺癌細胞萃取蛋白的專一性測試 82 圖二十四、Anti-hHb IgY對不同動物血紅素及heme proteins的專一性測試 84 圖二十五、Anti-hHb IgY與不同動物血紅素及heme proteins的反應程度比較 85 圖二十六、高溫處理對hHb抗原性之影響 88 圖二十七、胃蛋白酶水解人類血紅素的結果 90 圖二十八、胃蛋白酶水解處理對hHb抗原性影響的測試 91 圖二十九、兩種anti-hHb IgY樣品的純度與活性比較 95 圖三十、以雞隻生產Anti-hHb IgY抗體的建議方式 108 圖三十一、專一性抗體的萃取與吸附純化流程圖 110 圖三十二、應用hHb抗原管柱純化anti-hHb IgY時可能發生的問題 110 圖三十三、兔與雞的免疫注射與抗體收集過程的比較 119 表目錄 表一、民國97101年台灣的前五大死亡原因 5 表二、民國97101年台灣前五名惡性腫瘤資料 5 表三、四種大腸癌篩檢方式的優、缺點比較 10 表四、兩種糞便潛血檢測方法的優、缺點比較 17 表五、IgY與IgG的分子特性比較 26 表六、七種脊椎動物的血紅素胺基酸序列比對結果 29 表七、兔與雞在抗體生產與應用上之優缺點比較 30 表八、IgY抗體的部分應用案例 33 表九、四組雞隻血清的抗hHb效價倍數變化情形 50 表十、四組雞隻蛋黃的抗hHb效價倍數變化情形 52 表十一、一號抗原管柱的anti-hHb IgY純化結果 64 表十二、二號抗原管柱的anti-hHb IgY純化結果 73 表十三、雞隻生產專一性IgY的案例 101 表十四、雞隻蛋黃專一性抗體的比例 116 表十五、以兔或雞為抗體生產宿主,可得專一性抗體產量的比較 119

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