跳到主要內容

臺灣博碩士論文加值系統

(3.237.6.124) 您好!臺灣時間:2021/07/24 03:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:金鐸
研究生(外文):To Chin
論文名稱:刀豆球蛋白A(Concanavalin A)與醣蛋白結合的特性與檢驗應用
論文名稱(外文):Properties of Concanavalin A-glycoprotein binding and its clinical applications
指導教授:陳媛孃陳媛孃引用關係
指導教授(外文):Yuan-Liang Chen
學位類別:碩士
校院名稱:元培科技大學
系所名稱:醫學檢驗生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:99
語文別:中文
論文頁數:57
中文關鍵詞:酵素連結凝集素分析法刀豆球蛋白凝集素金膠濾泡激素
外文關鍵詞:ELLSAConcanavalin ALectincolloid goldFSH
相關次數:
  • 被引用被引用:0
  • 點閱點閱:1852
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
凝集素的應用主要是依靠與醣蛋白結合的專一性,刀豆球蛋白A是最早發現,也是研究應用最多的凝集素,大部分特性都是已知,所以最適合用來實驗新的測試方法。一般凝集素特性研究常使用酵素連結凝集素吸附檢測(ELLSA)來分析凝集素對醣蛋白的結合專一性,本研究第一部份利用刀豆球蛋白A來分析ELLSA進行過程中環境因素的影響,實驗結果發現ELLSA並不是在凝集素吸附醣蛋白飽和的狀態下,且會受光與熱影響,造成短時間非特異性結合升高,長時間則分解。奈米金膠是直接染色的技術,比酵素連結法快速,但是仍然有良好的靈敏度,本研究第二部份使用奈米金膠與刀豆球蛋白A結合,製作出刀豆球蛋白A的免疫金膠,並提出一種利用免疫金膠的新型凝集素特性測試法,與ELLSA進行比較,利用三種醣蛋白實驗的結果顯示,新方法同樣具有定量效果。濾泡促進激素也是一種醣蛋白,依照與刀豆球蛋白A的親和性分成三種分型,分型的比率與不孕症、癌症、腺體疾病等疾病有關,本研究最後部份製作出濾泡促進激素抗體的免疫金膠,以及利用刀豆球蛋白A的沈澱特性,提出新的濾泡促進激素的分型定量測試法,與ELLSA的結果相比較,實驗結果兩種方法部份趨勢符合,但該方法精確度還需更多實驗確認。
Lectin is used as a tool in identification and measurement of glycoproteins. Concanavalin A(Con.A) is the first lectin found, therefore it is most studied and best choice for modeling novel protocols. To study the specificity of a lectin, Enzyme-Linked Lectinosorbent Assay(ELLSA) is usually applied. In the first part of present research, the effect of environmental conditions to ELLSA is evaluated. The result showed lectin is not in saturation state binding with glycoproteins, and heat or light will increase non-specific binding in short duration, but degrade the proteins in longer duration. Gold colloid is a direct marker for proteins, the protocol is much faster than Enzyme-Linked methods. In the second part of the research, immunogold of Con.A is used to create a novel method for lectin research. The result on 3 different glycoproteins showed ability of this method to detect quantity of glycoproteins. Follicle-stimulating Hormone(FSH) has 3 isotype identified by the specificity to Con.A, the ratio of isotypes is linked with many diseases. The last part of the research used immunogold of FSH antibody and precipitaion of Con.A in a novel method to detect isotypes of FSH, and compare with ELLSA method. The result showed similar trend of both methods, but need more samples to confirm.
第一章 前言 1
1.1 凝集素概論 1
1.1.1 刀豆球蛋白A(Concanavalin A) 2
1.1.2 醣結構的命名 2
1.1.3 醣蛋白樣品介紹 4
1.1.3.1 Mannan 4
1.1.3.2 alpha1 acid(AGP) 4
1.1.3.3 Ovine Salivary Glycoprotein(OSM) 5
1.1.4 凝集素在生物體內的作用 5
1.2 酵素連結凝集素吸附檢測(Enzyme Linked Lectinosorbent Assay, ELLSA) 6
1.2.1 ELLSA 與 ELISA 的比較 6
1.2.2 酵素連結反應的誤差因素 7
1.3 濾泡激素 7
1.3.1 濾泡激素在人體中的作用 7
1.3.2 濾泡激素與疾病檢測 8
1.4 免疫金膠層析法 9
1.5 研究目的 10
第二章 材料與方法 11
2.1 儀器及設備 11
2.2 耗材 12
2.3 藥品 13
2.3.1 藥品資料 13
2.3.2 Enzyme-Linked Lectinosorbent assay 試劑配製 14
2.3.3 金膠配置 15
2.4 研究方法 15
2.4.1 Enzyme-Linked Lectin Absorbent Assay 流程 15
2.4.1.1 ELLSA 結合活性測試 15
2.4.1.2 ELLSA 不同環境條件對醣蛋白與凝集素結合性的影響 17
2.4.1.3 ELLSA 檢測 FB/WB 之 FSH 17
2.4.2 快速西方點墨法流程 18
2.4.2.1 實驗用strip的製作 18
2.4.2.2 測試理想的金膠與 Con.A / FSH 抗體比率 19
2.4.2.3 測試理想的金膠pH值 19
2.4.2.4 Con.A 免疫金膠製備 20
2.4.2.5 Con.A 金膠與醣蛋白結合測試 20
2.4.2.6 FSH 抗體免疫金膠製備 20
2.4.2.7 FSH 定量反應流程 21
第三章 數據處理 22
3.1 ELLSA數據 22
3.2 金膠數據 22
3.3 圖表製作 22
圖表使用gnuplot繪製,使用的平滑曲線函數為csplines。 22
第四章 結果與討論 23
4.1 Con.A 的ELLSA特性部份 23
4.2 Con.A 免疫金膠製作部份 23
4.3 金膠strip使用部份 24
4.4 FSH mAb免疫金膠的製作 24
4.5 Con.A與醣蛋白結合實驗部份 24
4.6 FSH 的檢測部份 25
第五章 圖表 26
第六章 結論 52
第七章 參考資料 53
一、英文部份:
Abramoff, M. D., Magelhaes, P. J., & Ram, S. J. (2004). Image Processing with ImageJ. Biophotonics International, 11(7), 36-42.
Abushoufa, R. A., Talbot, J. A., Brownbill, K., Rafferty, B., Kane, J. W., & Robertson, W. R. (2000). The development of a sialic acid specific lectin-immunoassay for the measurement of human chorionic gonadotrophin glycoforms in serum and its application in normal and Down’s syndrome pregnancies. Clinical Endocrinology, 52(4), 499-508. doi:10.1046/j.1365-2265.2000.00968.x
Asteria, C., Persani, L., Ferrari, M., & Beck-Peccoz, P. (1997). Unique binding pattern to concanavalin A lectin of glycoprotein hormones alpha-subunit hypersecreted by non-functioning pituitary adenomas. European journal of endocrinology, 137(6), 709.
Batteiger, B., Newhallv, W., & Jones, R. (1982). The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. Journal of Immunological Methods, 55(3), 297-307. doi:10.1016/0022-1759(82)90089-8
Bennett, M., & Schmid, K. (1980). Immunosuppression by human plasma alpha 1-acid glycoprotein: importance of the carbohydrate moiety. Proceedings of the National Academy of Sciences, 77(10), 6109.
Campo, S., Ambao, V., Creus, S., Gottlieb, S., Fernandez Vera, G., Benencia, H., & Bergadá, C. (2007). Carbohydrate complexity and proportions of serum FSH isoforms in the male: lectin-based studies. Molecular and cellular endocrinology, 260, 197–204.
Connolly, S. E., & Benach, J. L. (2005). The versatile roles of antibodies in Borrelia infections. Nature Reviews. Microbiology, 3(5), 411-420. doi:10.1038/nrmicro1149
Creus, S. (2001). Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity. Molecular and Cellular Endocrinology, 174(1-2), 41-49. doi:10.1016/S0303-7207(00)00453-6
Creus, S., Pellizzari, E., Cigorraga, S. B., & Campo, S. (1996). FSH isoforms: bio and immuno-activities in post-menopausal and normal menstruating women. Clinical Endocrinology, 44(2), 181-189.
Dam, T. K. (2004). Thermodynamic, Kinetic, and Electron Microscopy Studies of Concanavalin A and Dioclea grandiflora Lectin Cross-linked with Synthetic Divalent Carbohydrates. Journal of Biological Chemistry, 280(10), 8640-8646. doi:10.1074/jbc.M412827200
Duk, M. (1994). The Biotin/Avidin-Mediated Microtiter Plate Lectin Assay with the Use of Chemically Modified Glycoprotein Ligand. Analytical Biochemistry, 221(2), 266-272. doi:10.1006/abio.1994.1410
Frens, G. (1973). Preparation of gold dispersions of varying particle size: controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat. Phys. Sci, 241, 20-22.
Frikha-Gargouri, O., Gdoura, R., Znazen, A., Ben Arab, N., Gargouri, J., Ben Jemaa, M., & Hammami, A. (2008). Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of Chlamydophila pneumoniae IgA antibodies. BMC Infectious Diseases, 8(1), 98. doi:10.1186/1471-2334-8-98
Geoghegan, W. D., & Ackerman, G. A. (1977). Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. Journal of Histochemistry & Cytochemistry, 25(11), 1187-1200. doi:10.1177/25.11.21217
Horisberger, M., & Rosset, J. (1977). Colloidal gold, a useful marker for transmission and scanning electron microscopy. Journal of Histochemistry & Cytochemistry, 25(4), 295.
Imre, T., Schlosser, G., Pocsfalvi, G., Siciliano, R., Molnár-Szöllősi, É., Kremmer, T., Malorni, A., et al. (2005). Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography—electrospray mass spectrometry. Journal of Mass Spectrometry, 40(11), 1472-1483. doi:10.1002/jms.938
Jessen, H., & Behnke, O. (1986). Selective Binding of Colloidal Gold-Protein Conjugates to Epidermal Phosphorus-Rich Keratohyaline Granules and Cornified Cells. Journal of Investigative Dermatology, 87(6), 737-740. doi:10.1111/1523-1747.ep12456866
Jones, G. H., & Ballou, C. E. (1969). Studies on the structure of yeast mannan. I. Purification and some properties of an alpha-mannosidase from an arthrobacter species. The Journal of Biological Chemistry, 244(3), 1043-1051.
Koop, H. M., Valentijn-Benz, M., Nieuw Amerongen, A. V., Roukema, P. A., & Graaff, J. (1990). Aggregation of oral bacteria by human salivary mucins in comparison to salivary and gastric mucins of animal origin. Antonie van Leeuwenhoek, 58(4), 255–263.
Larsen, K., Thygesen, M., Guillaumie, F., Willats, W., & Jensen, K. (2006). Solid-phase chemical tools for glycobiology. Carbohydrate Research, 341(10), 1209-1234. doi:10.1016/j.carres.2006.04.045
Leifke, E., Simoni, M., Kamischke, A., Gromoll, J., Bergmann, M., & Nieschlag, E. (1997). Does the gonadotrophic axis play a role in the pathogenesis of Sertoli-cell-only syndrome? International Journal of Andrology, 20(1), 29-36. doi:10.1046/j.1365-2605.1997.00102.x
Liu, F. T. (2000). Galectins: a new family of regulators of inflammation. Clinical Immunology (Orlando, Fla.), 97(2), 79-88. doi:10.1006/clim.2000.4912
Loreti, N., Ambao, V., Juliato, C. T., Machado, C., Bahamondes, L., & Campo, S. (2009). Carbohydrate complexity and proportion of serum FSH isoforms reflect pituitary–ovarian activity in perimenopausal women and depot medroxyprogesterone acetate users. Clinical endocrinology, 71(4), 558–565.
Ngeh, J., Gupta, S., & Goodbourn, C. (2004). The reproducibility of an enzyme-linked immunosorbent assay for detection of Chlamydia pneumoniae-specific antibodies. Clinical Microbiology and Infection, 10(2), 171-174. doi:10.1111/j.1469-0691.2004.00757.x
Raffel, C., & Sell, S. (1982). The relationship between endocytosis of concanavalin A and phytohaemagglutinin receptors and blast transformation, and direct identification of individual rabbit lymphocytes reactive to both mitogens. Journal of Cell Science, 56, 141-156.
Rozwarski, D. A. (1998). Crystal Structure of the Lectin from Dioclea grandiflora Complexed with Core Trimannoside of Asparagine-linked Carbohydrates. Journal of Biological Chemistry, 273(49), 32818-32825. doi:10.1074/jbc.273.49.32818
SEFTALIOGLU, A., & DALÇIK, H. (1994). Immune Electron Microscopy. Journal of Islamic Academy of Sciences, 7(2), 120–129.
Shi, C., Zhao, S., Zhang, K., Hong, G., & Zhu, Z. (2008). Preparation of colloidal gold immunochromatography strip for detection of methamidophos residue. Journal of Environmental Sciences, 20(11), 1392–1397.
Sinha, S., Mitra, N., Kumar, G., Bajaj, K., & Surolia, A. (2005). Unfolding studies on soybean agglutinin and concanavalin a tetramers: a comparative account. Biophysical journal, 88(2), 1300–1310.
Sumner, J. B. (1919). The globulins of the jack bean, Canavalia ensiformis. Journal of Biological Chemistry, 37(1), 137.
Tammemagi, M. (1995). Methodological issues in assessing reproducibility—A comparative study of various indices of reproducibility applied to repeat ELISA serologic tests for Lyme disease. Journal of Clinical Epidemiology, 48(9), 1123-1132. doi:10.1016/0895-4356(94)00243-J
Ulloa-Aguirre, A. (1998). Structure-function relationship of follicle-stimulating hormone and its receptor. Human Reproduction Update, 4(3), 260-283. doi:10.1093/humupd/4.3.260
Vimr, E., & Lichtensteiger, C. (2002). To sialylate, or not to sialylate: that is the question. Trends in Microbiology, 10(6), 254-257. doi:10.1016/S0966-842X(02)02361-2
Wu, A. M., Lisowska, E., Duk, Maria, & Yang, Z. (2008). Lectins as tools in glycoconjugate research. Glycoconjugate Journal, 26(8), 899-913. doi:10.1007/s10719-008-9119-7
Wu, A. M., Wu, J. H., Herp, A., & Liu, J.-H. (2003). Effect of polyvalencies of glycotopes on the binding of a lectin from the edible mushroom, Agaricus bisporus. Biochemical Journal, 371(2), 311. doi:10.1042/BJ20021361
Yi, S. M., Harson, R. E., Zabner, J., & Welsh, M. J. (2001). Lectin binding and endocytosis at the apical surface of human airway epithelia. Gene Therapy, 8(24), 1826-1832. doi:10.1038/sj.gt.3301598
Zand, R., Agrawal, B. B. L., & Goldstein, I. J. (1971). pH-dependent conformational changes of concanavalin A. Proceedings of the National Academy of Sciences of the United States of America, 68(9), 2173.

二、中文部份:
吳明道. (1998). 淺談Gal 和GalNAc有特異性的應用凝集素(Applied lectins)對醣類結合性質的表達和分類. 生命科學簡訊, 12(1), 6.
吳芊槿. (2007). 以凝集化學法探討純化於刀豆中的刀豆球蛋白 A(Concanavalin A)與來源不同的醣蛋白結合能力. Taiwan: 正修科技大學.
蔡明松, & 吳千槿. (2010). 比較兩種甘露糖/葡萄糖專一性之刀豆凝集素及碗豆凝集素的結合性質. 化學, 28(3), 235-246.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top