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研究生:呂國豪
研究生(外文):Kuo-Hao Lu
論文名稱:藉由抗原決定部位印刷之高分子來去除血清中的高豐度蛋白
論文名稱(外文):Depletion of abundant proteins in serum using epitope-mediated imprinted polymers
指導教授:戴達夫戴達夫引用關係
指導教授(外文):Dar-Fu Tai
學位類別:碩士
校院名稱:國立東華大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:68
中文關鍵詞:白蛋白免疫球蛋白G分子印刷高分子
外文關鍵詞:albuminimmunoglobulin GMolecularly imprinted polymers
相關次數:
  • 被引用被引用:4
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  • 收藏至我的研究室書目清單書目收藏:1
摘要
人類血清白蛋白與免疫球蛋白G是血清中最高豐度的兩種蛋白。這些蛋白的存在造成血清蛋白質體分析時,檢體的注入量降低。將這些高豐度蛋白選擇性的移除能增加偵測的靈敏度。
分子印刷高分子是一種藉由模版誘導所構成具有專一辨識性的人工抗體。利用兩個十一個胺基酸長度的胜肽作為模版,製造出能夠吸附人類血清白蛋白與免疫球蛋白G的材料。這兩個胜肽分別為白蛋白與免疫球蛋白G的抗原決定部位。將形成的分子印刷高分子架構在石英晶體為天平上,利用這個測量方式判斷所選擇胜肽其序列與長度是否適合用於製造能吸附蛋白分子印刷高分子上。結果顯示出,天然的抗體是可被人工抗體取代的。
接著,將丙烯酸、丙烯醯胺、丙烯醯酪胺、乙基雙丙烯醯胺為單體之抗原決定部位印刷高分子嫁接到濾紙上做微量萃取。最後,將人工抗體嫁接到纖維素顆粒上,同時製備成一管柱用於大量去除高豐度蛋白。嫁接到纖維素上的人工抗體雖然其吸引力不如在石英微量天平上,但是更具實用性。一克的架接分子印刷高分子的纖維素顆粒,分別能夠吸附15.03毫克的白蛋白與10.01毫克免疫球蛋白G。
在臨床試驗中,100毫克的抗白蛋白印刷高分子纖維顆粒與50毫克的抗免疫球蛋白G印刷高分子纖維顆粒可以專一性地移除15 微升血清中83.5%的白蛋白與58.5%的免疫球蛋白G。
ABSTRACT
Human serum albumin (HSA) and immunoglobulin G (IgG) are the most abundant proteins in serum. The existence of these proteins decreases the sample loading capacity in analytic method of serum proteomics. Removal of abundant proteins alone should increase the sensitivity of detection.
Molecularly imprinted polymers (MIPs) are template-induced artificial antibodies with the ability to recognize and specifically bind the target molecule. We constructed a novel HSA and IgG binding material using two undecanpeptides as templates. These two undecanpeptides were the linear epitope of HSA and IgG. The epitope-mediated MIPs were tested at ng scale using quartz-crystal microbalance (QCM) protocol to evaluate whether the chosen sequence and length of peptides is suitable to construct MIPs binding site for the target protein. Our results demonstrated that antibody is replaceable.
Subsequently, the epitope-imprinted copolymer of acrylic acid, acrylamide, N-acryltyramine, and N,N’-ethylene bisacrylamide (EBAA) were implemented to graft cellulosic filter paper for microscale extraction. Finally, such artificial antibodies were fabricated onto cellulosic fiber and packed as a column for preparative depletion of the high-abundance protein. The affinity of these artificial antibodies on cellulose are not as specific as QCM chip but far more applicable. One gram of these cellulosic fibers was able to capture 15.03 mg of HSA or 10.01 mg of IgG, respectively.
In clinical trial, the 100 mg anti-HSA MIP-fiber and 50 mg anti-IgG MIP-fiber could specifically remove 83.5 % HSA and 58.5 % IgG in 15 μL serum.
TABLE OF CONTENTS

ACKNOWLEGEMENT………………………………………………..i
TABLE OF CONTENTS……………………………………………….ii
LIST OF TABLES…………………………………………………........v
LIST OF FIGURES……………………………………………….........vi
LIST OF ABBREVIATIONS………………………………………...viii
ABSTRACT………………………………………………………….....ix
ABSTRACT (in Chinese)…………………………………………….....x

1. INTRODUCTION…………………………………………………...1
1.1 MIPs as artificial antibodies………………………………...1
1.2 Scope of this work…………………………………………..2
1.3 Procedure of grafting MIP onto matrices…………………...3
2. BACKGROUND……………………………………………………..4
2.1 Molecularly imprinted polymer…………………………….4
2.1.1 Introduction…………………………………………..4
2.1.2 Molecular imprinting strategies and procedures……..5
2.1.3 Molecular interactions involved in non-covalent imprinting…………………………………………….7
2.1.4 Functional monomers………………………………...8
2.1.5 Polymerization procedure…………………………..10
2.1.6 Different MIP preparation method………………….11
2.2 Molecularly imprinted polymers in QCM…………………13
2.3 Serum for proteomic biomarker discovery………………..15
2.3.1 Introduction…………………………………………15
2.3.2 Biomarker Discovery……………………………….15
2.3.3 Major Protein Depletion Strategies…………………19
2.4 Molecular imprinting of protein…………………………...20
2.4.1 Introduction…………………………………………20
2.4.2 Obstacles in molecular imprinting of proteins……...20
2.4.3 Epitope approach for recognition of protein………..21
3. EXPERIMENTAL………………………………………………….23
3.1 Reagents and equipments………………………………….23
3.2 Equipments…………………………………………….......23
3.3 Preparation of imprinted polymer -coated QCM………….24
3.4 Evaluation of the frequency shifts of QCM……………….24
3.5 Synthesis of N-acryl tyramine……………………………..25
3.6 Fiber treatment and adsorption isotherm…………………..31
3.7 Coating MIP on Cellulosic Filter Paper…………………...31
3.8 Coating MIP on Cellulosic Fiber and the target molecule adsorption during filtration………………………………..32
3.9 Depletion of HSA and IgG from serum…………………...33
3.10 Two-dimension gel electrophoresis……………………….33
3.11 Analysis of depletion percentage of abundant proteins in serum………………………………………………………34
4. RESULT AND DISCUSSION……………………………………...35
4.1 Fabrication of MIP on QCM chip to detect HSA and IgG...35
4.1.1 To determine the epitope by QCM assay…………...35
4.1.2 The binding effect of MIP-QCM depends on the length of epitope peptide as template and the size of protein………………………………………………41
4.2 Synthetic artificial antibody grafted onto cellulosic matrix.46
4.2.1 Formation of self-assembly monolayer of MPS on the surface of cellulose……………………………...46
4.2.2 Synthetic artificial antibody grafted onto cellulosic filter paper to bind HSA and IgG…………………...48
4.2.3 Synthetic artificial antibody grafted onto cellulosic fiber to bind HSA and IgG………………………….51
4.3 Depletion of abundant proteins in serum using epitope -mediated imprinted polymer……………………………...60
4.3.1 Improvement the resolution of serum proteins in 2D gel after HSA and IgG depletion……………………60
4.3.2 Evaluation of the efficiency of depletion by HPLC...62
5. CONCLUSION……………………………………………………..64
6. REFERENCE………………………………………………………65
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