臺灣博碩士論文加值系統

麩胺酸草醯乙酸轉胺酶 (glutarmate oxaloacetate transaminase，GOT)，又稱天門冬胺酸轉胺酶( Aspartate amino Transferase，AST )。GOT廣存於各細胞組織，如心臟、肝臟及肌肉組織，當上述組織受到損傷GOT會釋放到血清。所以GOT檢測已應用於診斷有關心、肝及膽方面的疾病。但GOT於體外的半衰期為17小時，如以酵素法無法完全得到真正GOT值。本研究主要在進行GOT之單株抗體之產製與純化及檢測GOT之酵素連結免疫吸附分析法(ELISA)之分析系統建構。
首先進行GOT之單株抗體之生產與純化。以TiterMax佐劑乳化GOT後行尾巴注射免疫。免疫後一個月後進行實施追加注射，2~3週後進行細胞融合。經HAT培養液及間接三明治ELISA法篩選，共得到6株分泌高效價抗GOT抗體的融合瘤細胞株(hybridoma)，經限數稀釋、擴大培養後。其抗體亞型（Isotype）經鑑定為IgG1重鏈，k輕鏈。挑選效價最高的兩株1P8F與2P7B進行抗體生產。經由小鼠腹腔培養法大量生產單株抗體後，以50 %硫酸銨將抗體沉澱。經透析，再以DEAE離子交換層析法純化抗體。純化之單株抗體特異性高稀釋至57仍然有抗體反應。抗體以PBS透析後，冷凍保存。
以雙功能連結試劑法（bifunctional closslinker reagent）將HRP（Horse radish peroxidase）與抗體連結形成酶標記抗體，將抗體稀釋100000倍後仍有反應。並將其應用至直接型ELISA法偵測GOT。此酶標記物稀釋至8000~10000倍後，可得GOT濃度範圍於0~2 mg/ml時與吸光值(OD 410nm)呈現一線性正比關係。

GOT, glutarmate oxaloacetate transaminase, AST L-aspart transaminase ,is a normal enzyme in the tissue of heart 、liver and muscle of human. When the tissue of liver、heart and muscle are hurt, GOT will release to the blood. With the character of GOT ,we can check the damage of heart、liver and muscle. Our research focus on the production and purification of Monoclonal Antibody to against GOT, and construct a ELISA system to detect GOT with the MAb.
For immune the mouse, we emulsify GOT with TiterMax, and inject the emulsion to the button of the mouse’s tail. After a month, boost it with GOT. After 2~3 weeks, fuse the spleen cell of the immuned mouse and the myaloma cell. After screen by ELISA, we got 6 hybridomas secret high-titer monoclonal antibody. The Mabs all have g heavy chain and k light chain. We choice two main MABs（2P7B、1P8F）be the main cell line to product antibody. In the 50% Ammonium sulfate most antibody will precipite, and follow with DEAE ion exchange chromatography to get pure antibody. After dialysis in PBS, store it in powder form in -20℃.
Conjugate IgG and HRP with bifunctional crosslinker reagent（SMCC） to product HRP-Ab, The HRP-Ab could detect 5 mg/ml GOT coating on ELISA plate, after dilute 100000 times. So we can construct a direct ELISA system to detect GOT. When dilute the HRP-Ab 8000~10000 times , we can detect 0~2 g/ml GOT exactly.

目錄 I
中文摘要 Ⅲ
英文摘要 Ⅳ
第一章 研究背景 .
第一節 GOT簡介與傳統檢測方式
(一)、 GOT之簡介 1
(二)、 傳統GOT檢測方式結果與討論 3
第二節 單株抗體與融合瘤技術 .
(一)、 抗體結構 5
(二)、 抗體特性 8
(三)、 多株抗體 9
(四)、 單株抗體 9
(五)、 單株抗體的製備 10
1. 動物免疫 10
2. 融合瘤細胞之篩選 13
3. 篩選與單株化 14
4. 哺乳動物細胞培養 18
5. 單株抗體之生產與純化 19
第三節 酵素連結免疫分析法原理
（一） 原理
（二） 抗體之標記
1. 過碘酸鈉法 24
2. 雙功能連結試劑法 26
第二章 材料與方法 .
一. 免疫分析方法 29
二. 眼窩採血 32
三. 小鼠免疫 34
四. 骨髓癌細胞培養 41
五. 細胞融合 42
六. 擴大培養 44
七. 限數稀釋 45
八. 細胞冷凍與解凍 47
九. 單株抗體大量生產 49
十. 單株抗體class與subclass種類之鑑定 50
十一. 多株抗體生產 51
十二. 抗體純化 52
十三. 蛋白質定量 54
十四. 酶標記物之製備 55
第三章 結果與討論 .
第一節 單株抗體的生產與篩選 57
一. 篩選系統建立 57
(一)、 採血 57
(二)、 ELISA偵測抗體 58
二. 融合細胞株之選擇 58
三. 小鼠免疫 59
四. 不同免疫法之比較 60
五. 融合條件探討 65
六. 細胞株之篩選 65
七. 擴大培養 70
八. 單株化 70
九. 單株抗體亞型之鑑定 71
十. 單株抗體之純化 71
第二節 建立偵測GOT之系統 .
一. 酶標記物之製備 76
二. 以直接型ELISA檢測GOT 78
第四章 結論 81
第五章 參考資料 82

roblewski, F., and La Due, J.S.：Serum glutamic oxaloacetic transaminase activity and a index of lieu cell injary, A preliminary report Ann Int Med 43:345-360, 1995.
2. 吳昭新,李元成：肝功能檢查（二）GOT及GPT , 當代醫學,4：1977,1936-1941.
3. Reittmen, S. and Frankel, S.：A colormetric method for the determination of serum glutamic oxalacetic and glutarmic pyruvate transaminase ,Am. J. Clin Path., 28：56-63,1957
4. Clermont, RJ and Chalmer, J.C.：The transaminase test in liver disease Medicine, 46：197~207, .
5. Karmen , et al,1955
6. Price CP, Alberti KGMM. Biochemical assessment of liver function. In wright R, Alberti KGMM, Karran S, Milward-Sadler GH, Editor. Liver and biliary disease. London：WB saunders, 1979;381-416
7. 何敏夫，臨床酵素學,臨床化學,1994,442-447.
8. Golding, J.W., Antibody Production by hybridoma. J. Immunol. Methods, 1980,39:285-308,.
9. Books D., and Zola, H., Technique for production and Characterization of Monoclonal Hybridoma Antibody：Techniques and Application, CRC PRESS: 1-58,1982.
10. Golding, J.W., Monoclonal Antibodies ： Principle and practice , production and application of monoclonal antibodies in Cell Biology, Biochemistry and Immunology, Academic Press, Inc.,1983.
11. Price, P. J., Hybridoma Technology, in Advances in cell culture. 4：157~177, 1985.
12. Kohler, G. and Milstein, C.（1975）.Fusion of two immunoglobulin-production myeloma cells. Nature 256, 495-497
13. Burrent, F. M.（1957）.A modification of Jerne’s theory of antibody production using the concept of clonal selection . Austral. J. Sci. 20,67-69
14. Cotton, R.G.H. and Milstein, C.（1973）.Fusion of two immunoglobulin-producing myeloma cells. Nature 244, 42-43
15. Hefle, S.L., Immunoassay fundamentals. Food Technol., 102-107,1995
16. Siklodu, B., Barna-Vetro, I., and Solti, L., Latent Immunization to produce High-Affinity Monoclonal Antibodies to Pregesterone, Hybridoma, 14（1）:79-84,1995
17. John R. Crowther.2001.System in ELISA .The ELISA Guidebook, 19-22
18. 莊榮輝，水稻蔗糖合成酶之研究，國立台灣大學農業化學研究所博士論文，台北市（1985）
19. Paul K., Nakane., and Arika Kawaoi, Peroxidase-labling Antibody a new method of conjugate,1974,J. Histochemistry Cytochemistry, 1084-1091
20. Makoto Nishimura, et al., Development and evalution of a direct sandwich — enzyme-linked immunosorbent assay for the quantification of human hepatic triflyceride lipase mass in human plasma., J. Immuno. Method. , 2000,41-51.
21. P. Tijssen and E. Kurstak, Highly Efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody Conjugate for Enzyme immunoassays.,1984, Anal. Biochem. 136, 451-457
22. Water. P.M., et al,2000, Characteristation of monoclonal antibody to ovine interleukin-6 and development of sensitive capture ELISA, Vete. Immuno. Immunopathology., 155-165.
23. Review 10 June 1971, Communication To the Editor, Immunochemistry, Pergamon Press., 8：1175-1179
24. Boorsma. D.M., and Kalsbeek. G.L., A comparative study of horseradish peroxidase conjugate prepare with a one-step and a two step method., 1975,J. Histochem. Cytochem., 23（3）:200-207.
25. Hashida. S., Imagawa. M., et al., More useful maleimide compounds for the conjugation of Fab’s to horseradish peroxidase through thiol group in the hinge. J. Appl. Biochem., 1984,6,56-63.
26. Yoshitake. S., Yamada. Y., Ishikawa. E., et al., Conjugation of glucose oxidase from Aspergillu. Niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide . Eur. J. Biochem. 1979, 101,395-399.
27. Brinkley. M.A.,（1992）.A survey of methods for preparing protein conjugates with dyes, haptens and crosslinker reagent . Bioconjugate chem., 3,2-13.
28. Partis. M.D., Griffiths, D.C. and Beechey. R.B.（1983）. Crosslinking of proteins by omega-maleimido alkanoyl N-hydroxysuccinimide ester. J. Protein. Chem. 2,263-277.
29. 賴昭伶，氯酶素單元抗體之產製及其應用於壓電免疫感測器之研究，國立台灣大學農業化學研究所碩士論文，台北市（1997）
30. 周淑芬，多功能免疫反應型腫瘤標記感測器之開發研究，國立台灣大學農業化學研究所碩士論文，台北市（2000）
31. 徐錫敏，L-麩胺酸氧化酶單株抗體之製備與鑑定，國立台灣大學農業化學研究所碩士論文，台北市（1988）
32. Peters J. H., Baumgarten H.(Eds), Taking blood from mice,, Monoclonal Antibodies，1992,New York,p71-73
33. Zola Z, Immunization, Monoclonal Antibody,2001,UK, p21-P23.
34. Peters J. H., Baumgarten H.(Eds), Immunnizing Mice,, Monoclonal Antibodies，1992,New York,p47-49
35. Roberge, F.G., Xu, D. and Chan, C.C. A new effective and n0n-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis. Current Eye Resesrch , 11:371-376, 1992
36. Hunter, R. L., Olsen , M.R. and Bennett, B. Copolymer Adjuvant and TiterMax® . In The Theory and practical Application of adjuvants. D.E.S. Stewart-Tull, editor. John Wiley & Sons, New York,pp51-94, 1995
37. Benntt, B. et al.,Acomparsion of commercially available adjuvants for use in research. Joural of immunological Methods 153:31-40, 1992.
38. Leenaars M., et al., Immune responses and side effect of five different oil-based adjuvants in mice., Veterinary Immuno. Immunopathology, 61,（1998）,291-304.
39. D. L. Harlon, Ed., Antibodies: A laboratory Manual, Cold Spring Habor Laboratory, New York, U.S.A.（1988）
40. Peters J. H., Baumgarten H.(Eds), Protein A column Chromatography, Monoclonal Antibodies，1992, New York, pp264 — 270.
41. D. L. Harlon, Ed., Antibodies: A laboratory Manual, Cold Spring Habor Laboratory, New York, U.S.A.（1988）, p306-311
42. Golding, J.W., Monoclonal Antibodies ： Principle and practice , production and application of monoclonal antibodies in Cell Biology, Biochemistry and Immunology, Academic Press, Inc.,1983.p105-116
43. Zola Z, Immunization, Monoclonal Antibody,2001, UK, p42
44. 莊榮輝等，基本分析方法，酵素化學實驗，國立台灣大學農業化學系 生物化學研究室，2000,p193
45. Tresca J.P., Ricoux R., Pontet M., Engler R., Comparative activity of peroxidase-antibody conjugate with periodate and glutaraldehyde coupling according to an enzyme immunoassay. , Ann Biol clin , 1995, 53:227-231
46. Gosling J. P., Immunoassay
47. Hunter, R.L., Olsen, M.R., and Bennett, B., Copolymer adjuvant and Titer Max, in The Theory and Practical Application of Adjuvant, Stewar-Tull, D.E. S., Ed., Wiley, New York,1995,51.
48. Harlon, D. L. Ed, Antibodies: A laboratory Manial, Cold Spring Harbor Laboratory, New York, U.S.A. (1988)
49. 曾怡聲，腫瘤標記 Sialyl Lewis X單源抗體之產製及其應用於壓電免疫感測氣之開發研究，P38，台北市(2001)