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研究生:吳佳容
研究生(外文):Wu Chia-Jung
論文名稱:海鱺免疫球蛋白之萃取及其酵素結合免疫吸附檢測法發展之應用
論文名稱(外文):Isolation of immunoglobulin from Cobia (Rachycentron canadum) and its application for the develpoment of ELISA test kit
指導教授:楊惠郎陳世輝陳世輝引用關係
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
論文頁數:86
中文關鍵詞:海鱺免疫球蛋白酵素結合免疫吸附檢測法
外文關鍵詞:CobiaimmunoglobulinELISA
相關次數:
  • 被引用被引用:4
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  • 下載下載:158
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摘 要
海鱺(Rachycentron canadum)係我國最近才成功完成人工養殖的魚種,是我國政府及業界選為推動箱網養殖產業,為進軍國際市場之主力魚種。但養殖不至三年海鱺之存活率卻急速下降,依病魚解剖一般咸信死亡原因以傳染疾病為主,故研發海鱺反應免疫之酵素結合免疫吸附檢測系統(ELISA)以協助海鱺之疾病防治。
本論文的實驗首先抽取海鱺血清,經45-50%硫酸銨沈澱及分子篩管柱層析法(gel filtration chromatography)的篩選,分離出海鱺免疫球蛋白(Ig)。並以還原態及非還原態聚丙烯氨凝膠電泳(reduced SDS-PAGE and non-reduced SDS-PAGE)評估其分子量,確定此抗體的重鏈(heavy chain)及輕鏈(light chain)之分子量各約75 kDa及30 kDa;其IgM的單體(monomer)分子量約為190 kDa,又因其聚合體(polymer)之分子量為670 kDa,我們初步認為其為四體(tetramer)的結構。將海鱺抗體多次免疫注射白兔後以製備二級抗體(rabbit anti-Cobia IgG),以蛋白質A吸附性層析管柱(Protein A Sepharose)純化二級抗體,再以免疫轉漬分析(immunoblotting)測試其專一性,此二級抗體對海鱺Ig具有特異性的結合,對人類IgM則無反應,其抗體強度則經ELISA測試後顯示,此二級抗體稀釋倍數可以高達1:32,000,在已具專一性及高抗體力價之條件下發展酵素連結免疫吸附法。我們使用海鱺之陰性及陽性對照組血清棋盤式方格法,經多次實驗,以使用下列條件為套組之最適條件:每孔洞使用20μg之抗原蛋白,檢測血清稀釋濃度為1:50倍,二級抗體稀釋1:10,000倍。依以上條件應用於弧菌海鱺人工免疫效果檢測,得知施打疫苗組與未施打疫苗組魚隻的血清吸光值約有四倍的差異,證明此一方法可以使用於偵測海鱺血清中特異抗體的存在,並有可靠之重複性、理想之特異性及敏感性。期盼未來可將本試劑應用於海鱺流行病學的調查。
Cobia (Rachycentron canadum) has been completely farmed recently with great success in Taiwan. It is selected by the aquaculture industry as the promising fish for cage aquaculture and target to be promoted to international market. However, after couple years of farming the survival rate decreased drastically. It is to believe that the causes of mortality are infectious diseases of bacterial source. To improve the prophylaxis and treatment of Cobia disease, a simple diagnostic method such as ELISA which can test Cobia immunization response would be desirable.
To develop ELISA test, a Cobia Ig was isolated by 45-50% ammonium sulfate precipitation and by gel filtration chromatography. The molecular weight of the Ig was estimated by reduced SDS-PAGE and non-reduced SDS-PAGE. The heavy chain and light chain of the Cobia Ig are found to be 75 kDa and 30 kDa, respectively. Since the molecular weight of the Cobia Ig monomer is about 190 kDa and the polymer is 670 kDa, the structure of the Cobia Ig was proposed to be tetramer. The Cobia Ig was injected into rabbit several times to induce rabbit anti-Cobia IgG. The secondary antibody was purified by Protein A Sepharose. As testing the specificity by immunoblotting, the rabbit anti-Cobia antibody did not react with human IgM, and the antibody titer has reached 1:32,000 by ELISA test. With this secondary antibody, the microtiter plate ELISA kit was developed. The ELISA kit was examined repeatedly with positive and negative serum of Cobia.
The most appropriate condition for antibody assay was 20μg antigen in each well, 1:50 dilution of the serum, and 1:10,000 dilution of the secondary antibody. Using the same condition in artificial immunization and non-immunization models in Vibrio anguillarum, the difference of OD ratio was found at least four folds. This kit also provides a way that can be used for the detection of the existence of specific antibody in Cobia serum. We look forward to employ the kit for epidemic study of Cobia in the future.
目 錄
項目 頁數
中文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ I
英文摘要‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ III
致謝‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ V
目錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VII
圖表目錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ XI
縮寫及符號‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ XIII
壹、緒言‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
一、台灣水產養殖的概況及海鱺的細菌疾病‧‧‧‧‧‧‧‧‧ 1
二、魚類免疫球蛋白的研究‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5
三、酵素連結免疫吸附法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 9
四、研發ELISA套組於海鱺養殖疾病預防之功能‧‧‧‧‧‧‧‧ 11
貳、研究目的及實驗架構‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 13
參、材料與方法‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 14
一、弧菌之來源‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 14
二、弧菌之培養與蛋白質的萃取‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 14
三、海鱺養殖與血清抽取‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 15
四、海鱺血清免疫球蛋白之純化‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 16
五、海鱺免疫球蛋白還原態之聚丙烯胺膠體電泳
(reduceed SDS-PAGE) ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
16
六、海鱺免疫球蛋白非還原態之聚丙烯胺膠體電泳
(non-reduced SDS-PAGE) ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
17
七、海鱺免疫球蛋白之蛋白質濃度測定‧‧‧‧‧‧‧‧‧‧‧ 18
八、白兔抗海鱺抗體(rabbit anti-cobia antibody)製備‧‧‧‧‧‧ 19
九、純化白兔抗海鱺抗體IgG‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 19
十、使用還原態之聚丙烯胺膠體電泳(reduced SDS-PAGE)
分析rabbit anti-Cobia IgG‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
21
十一、免疫轉漬分析(immunoblotting) ‧‧‧‧‧‧‧‧‧‧‧ 21
十二、測試ELISA之非特異性結合‧‧‧‧‧‧‧‧‧‧‧‧‧ 22
十三、測定rabbit anti-Cobia IgG之適當稀釋倍數範圍‧‧‧‧‧ 24
十四、製備應用於棋盤式方格定量法標準陽性及陰性血清檢體‧ 25
十五、抗原抗體之棋盤式方格定量法
(checker-board titration) ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
25
十六、海鱺免疫注射弧菌疫苗後血清抗體力價分析‧‧‧‧‧‧ 26
十七、試劑應用性分析:海鱺血清樣本檢驗‧‧‧‧‧‧‧‧‧ 26
十八、實驗材料及來源‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 28
A、試劑配方‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 28
B、藥品‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 36
C、儀器‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 38
肆、結果‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 39
一、蛋白質濃度定量實驗之BSA標準曲線‧‧‧‧‧‧‧‧‧‧ 39
二、海鱺Ig及人類IgM之reduced SDS-PAGE電泳圖‧‧‧‧‧ 39
三、海鱺Ig之non-reduced SDS-PAGE電泳圖‧‧‧‧‧‧‧‧‧ 40
四、Rabbit anti-Cobia IgG的reduced SDS-PAGE電泳圖‧‧‧‧ 40
五、海鱺Ig及人類IgM之免疫轉漬分析結果‧‧‧‧‧‧‧ 41
六、測試ELISA套組之非特異性結合‧‧‧‧‧‧‧‧‧‧‧‧ 41
七、測試 rabbit anti-Cobia IgG之適當稀釋倍數‧‧‧‧‧‧‧‧ 42
八、製備應用於棋盤式方格稀釋實驗之陽性及陰性血清‧‧‧‧ 43
九、陽性及陰性血清棋盤式方格稀釋結果‧‧‧‧‧‧‧‧‧‧ 43
十、計算最佳抗原用量及抗體濃度‧‧‧‧‧‧‧‧‧‧‧‧‧ 44
十一、建議之ELISA操作程序‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 45
十二、免疫注射弧菌疫苗海鱺血清抗體力價之分析結果‧‧‧‧ 45
十三、海鱺血清樣本檢驗‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 46
十四、免疫前後與海鱺血清樣本檢驗兩者抗體力價之比較‧‧‧‧ 47
伍、討論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 48
一、海鱺的免疫球蛋白‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 48
二、兔抗血清的製備‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 51
三、建立海鱺ELISA抗體偵測系統‧‧‧‧‧‧‧‧‧‧‧‧‧ 53
四、海鱺ELISA抗體偵測系統的應用‧‧‧‧‧‧‧‧‧‧‧‧ 54
陸、結論‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 57
柒、參考文獻‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 58
捌、圖表‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 63
玖、附錄‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 81
拾、自述‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 85
拾壹、授權書‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 86
圖 目 錄
項目 頁數
圖一、使用BSA為檢體之蛋白質定量標準曲線‧‧‧‧‧‧‧‧ 63
圖二、海鱺Ig及人類IgM之reduced SDS-PAGE電泳比較圖‧‧ 64
圖三、海鱺Ig之non-reduced SDS-PAGE電泳圖‧‧‧‧‧‧‧‧ 65
圖四、rabbit anti-Cobia IgG之reduced SDS-PAGE電泳分析圖‧‧ 66
圖五、免疫轉漬分析(western blot)證明rabbit anti-cobia IgG
之專一性‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
67
圖六、測試ELISA套組之背景值‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 68
圖七、rabbit anti-Cobia IgG適當稀釋倍數之分析圖‧‧‧‧‧‧ 69
圖八、應用於棋盤式方格稀釋實驗之陽性及陰性血清之比較‧‧‧ 70
圖九、陽性血清棋盤式方格稀釋結果‧‧‧‧‧‧‧‧‧‧‧‧‧ 71
圖十、陰性血清棋盤式方格稀釋結果‧‧‧‧‧‧‧‧‧‧‧‧‧ 72
圖十一、陽性及陰性血清專一性分析結果之比較‧‧‧‧‧‧‧‧ 73
圖十二、免疫前後海鱺血清抗V. anguillarum O1 strain
之抗體力價‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧
74
圖十三、海鱺血清樣本檢驗(第1至17號血清)‧‧‧‧‧‧‧‧ 75
圖十四、海鱺血清樣本檢驗(第18至36號血清)‧‧‧‧‧‧‧ 76
圖十五、海鱺血清樣本檢驗(第37至52號血清)‧‧‧‧‧‧‧ 77
圖十六、免疫前後與海鱺血清樣本檢驗兩者抗體力價之比較‧‧‧ 78
表一、實驗架構流程圖‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 79
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Purification and partial characterization of serum immunoglobulin of European perch(Perca fluviatilis L.)
Fish and Shellfish Immunology 1993. 3:331-343
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