蛋白質 G 與具硫基反應性的釕複合物之生物接合作為螢光免疫試驗的通用試劑

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林璟棠(Jing-Tang Lin) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

蛋白質 G 與具硫基反應性的釕複合物之生物接合作為螢光免疫試驗的通用試劑
(A Sulfhydryl-Reactive Ruthenium (II) Complex and its Conjugation to Protein G as a Universal Reagent for Fluorescent Immunoassays)

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摘要(中)

為了在生物感測上發展螢光釕複合物，我們合成一個新穎的硫基反應化合物， 4-bromophenanthroline bis-2,2`-dipyridine Ruthenium bis (hexafluorophosphate) 。將完成的釕複合物接合至硫修飾的蛋白質 G 上形成螢光免疫試驗的通用試劑。並且透過SDS 凝膠電泳確認釕(II)-蛋白質 G 複合體的成功接合。釕(II)-蛋白質 G 複合體在452 奈米激發波長下可以得到602 奈米的放射波峰，表示釕(II)-蛋白質 G 複合體在完成接合後仍保有與釕複合物相似的螢光特性。為了測試接合物在生物感測上的可用性，我們執行免疫球蛋白 G 結合試驗。其結果表現出釕(II)-蛋白質 G 複合體有能力接至免疫球蛋白 G ，並且愈多交聯物修飾就有愈高的接合效率。在測定釕(II)-蛋白質 G 複合體在螢光免疫試驗的可行性中，發展出利用複合體與抗組胺酸抗體去偵測組胺酸標記蛋白的方法。結果顯示組胺酸標記蛋白在不同劑量被下成功的偵測出來，顯示出蛋白質 G 複合體可以當作免疫定量試驗中的通用螢光試劑。

摘要(英)

To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2`-dipyridine
Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

關鍵字(中)

★ 免疫試驗
★ 釕複合物
★ 蛋白質 G
★ 螢光

關鍵字(英)

★ Fluorescence
★ Protein G
★ Ruthenium (II) Complex
★ Immunoassays

論文目次

中文摘要 - I
ABSTRACT - II
ABBREVIATION - III
致謝 - IV
TABLE OF CONTENTS - V
LIST OF FIGURES - VII
I. INTRODUCTION - 1
I. 1. Biosensing - 1
I. 2. Immunoassay - 1
I.2.1. Immunoglobulin G - 7
I.2.2. Protein G - 7
I. 3. Fluorescence - 7
I. 4. Ru(II) polypyridine complex - 8
I. 5. Study Outline - 8
II. MATERIALS AND METHODS - 10
II. 1. Synthesis of 4-bromophenanthroline bis-2,2`-dipyridine Ruthenium bis (hexafluorophosphate) - 10
II. 2. Conjugation of Ru(II) complex and protein G - 10
II. 3. Absorbance and emission spectra measurement - 11
II. 4. Fluorescence decay curve - 12
II. 5. Mass spectra - 12
II. 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) - 12
II. 7. IgG binding assay using Ru(II)-protein G conjugates - 13
II. 8. pH effect of conjugation - 14
II. 9. Purification of recombinant histidine-tagged protein - 14
II. 10. Detection of recombinant histidine-tagged protein using Ru(II)-protein G conjugates - 15
III. RESULT AND DISCUSION - 17
III. 1. Feature of Ru(II) complex - 17
III. 2. Conjugation of protein G with Ru(II) complex - 23
III. 3. Identification of Ru(II)-protein G conjugates by SDS-PAGE - 25
III. 4. Identification of Ru(II)-protein G conjugates by ESI-Mass - 27
III. 5. Absorbance and emission spectra of Ru(II)-protein G conjugates - 35
III. 6. IgG-binding assay of Ru(II)-protein G conjugates - 37
III. 7. Application of Ru(II)-protein G conjugates in fluorescent immunoassay - 43
IV. CONCLUSION - 47
VI. REFERECES - 48
APPENDIXES: PLOS ONE PUBLICATION REPRINT - 51

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指導教授

陳健生、畢勒曼
(Chien-Sheng Chen、Jean-Francois Biellmann)

審核日期

2012-6-20

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