臺灣博碩士論文加值系統

酵素免疫吸附測定法(ELISA) 為一種利用抗原-抗體專一性結合之檢測工具，已廣泛應用於生物醫學以及各種產業領域，然而仍面臨抗體偵測敏感度不足的問題。為了解決此問題，我們開發一種新型Poly-protein G cell-based ELISA平臺，將穩定表現Poly-protein G之細胞固定於ELISA平臺，提升抗體固定於ELISA平臺之數量，進而提升偵測敏感性。功能性protein G或Poly-protein G可穩定表現於細胞上，且被protein G或Poly-protein G所抓取之anti-PEG antibody，仍保有其PEG辨認之專一性。Poly-protein G cell-based ELISA平臺所能承載之抗體量較傳統ELISA平臺高出之2至18倍。且Poly-protein G cell-based ELISA平臺確實可應用於sandwich ELISA，並對於各種PEG或PEG修飾物(PEG10K、PEG2K-Lipo-dox以及 PEGASYS)之偵測效率皆遠高於傳統ELISA平臺。重要的是，poly-protein G cell based ELISA平臺之偵測敏感度甚至可達 pg/ml之水平。因此poly-protein G cell based ELISA平臺可大幅提升抗體固定於ELISA平臺之數量，並保有抗體專一性及方向性，未來將可取代傳統ELISA平臺，應用於生物醫學及各領域之研究。

Enzyme-linked immunosorbent assay (ELISA) has been widely used as an analytical tool in medicine and in various biotechnological applications. However, low sensitivity is a weakness of this method. We have developed a novel poly-protein G cell-based ELISA plate based on fixing membrane poly-protein G expressing cell on ELISA plate; thereby increasing the coating amount and detective sensitivity of antibody. Functional　protein G or poly-protein G was stably expressed on cell membrane. The anti-PEG antibodies which were coated on poly-protein G cell-based ELISA plate was found to preserve PEG-binding activity. The antibody-coating capacity of the poly-protein G cell-based ELISA plate was 2−18 times higher than traditional ELISA plate. More importantly, the sensitivity for detecting PEG and pegylation molecular (PEG10K、PEG2K-Lipo-dox and PEGASYS) using anti-PEG antibodies coated on poly-protein G cell-based ELISA plate was significantly greater than traditional ELISA plate. In addition, the poly-protein G cell-based ELISA plate can be used to prepare a sandwich ELISA assay resulting in an increase in the coating amount of antibodies on ELISA plate; preserving the specificity and orientation of antibodies. Therefore, the poly-protein G cell-based ELISA plate ai a rald replacement for the traditional ELISA plate and is applicable in various industries.

標題 i
臺北醫學大學碩士學位考試委員審定書 ii
臺北醫學大學電子暨紙本學位論文書目同意公開申請書 iii
臺北醫學大學學位保密同意書暨簽到表 iv
誌謝 vi
目錄 vii
中文摘要 xi
英文摘要 xii
第一章 緒論
1.1 酵素免疫吸附測定法(簡稱ELISA)及其面臨之問題................................1
1.2 改良傳統ELISA平臺之 Antibody expressing cell based ELISA 平臺.......2
1.3 改良傳統ELISA平臺與 Antibody expressing cell based ELISA 平臺之新型Poly-protein G cell-based ELISA平臺........................................................3
第二章 研究材料與方法
2.1　藥品與試劑.........................................................................................5
2.2　細胞株................................................................................................6
2.3 建構poly-protein G基因之基因克隆試驗...............................................6
2.4 建構永久穩定表現poly-protein G之細胞株.............................................7
2.5 3T3/protein G與3T3/poly-protein G之膜蛋白表現與建構長度差異確認...............................................................................................................7
2.6 評估膜上protein G及poly-protein G抓取抗體之功能.............................8
2.7 分析被protein G或poly-protein G所抓取之抗體，其辨識抗原之功能.........8
2.8 分析傳統ELISA平臺之抗體承載量........................................................9
2.9 分析protein-G cell based ELISA plate以及poly-protein G cell based ELISA plate之抗體承載量.......................................................................................9
2.10 測試傳統ELISA plate對PEG5K-biotin之偵測敏感性............................10
2.11 測試protein-G cell based ELISA plate以及poly-protein G cell based ELISA plate對PEG5K-biotin之偵測敏感性..............................................................11
2.12 分析傳統ELISA plate應用於三明治酵素連結免疫吸附法( sandwich ELISA)時，對PEG10K 或PEG2K-Lipo-dox之偵測敏感性..............................12
2.13 Poly-protein G cell based ELISA plate應用於三明治酵素連結免疫吸附法( sandwich ELISA)並應用於測試PEG10K 或PEG2K-Lipo-dox.........................13
2.14 評估傳統 ELISA plate以不同濃度之capture antibody偵測PEG10K 或PEG2K-Lipo-dox之偵測效率........................................................................14
2.15 傳統 ELISA plate應用於測試不同濃度之PEG修飾蛋白藥物- (PEG-alpha interferon 2α;PEGASYS) .............................................................................15
2.16 Poly-protein G cell based ELISA plate應用於測試不同濃度之PEG修飾蛋白藥物- (PEG-alpha interferon 2α;PEGASYS) ................................................15
2.17 試驗5 % FBS (Fetal bovine serum)是否可將未承載抗體之poly-protein G完全封閉.......................................................................................................16
第三章 分析與結果
3.1　建構細胞膜上穩定表現protein G之細胞株...........................................18
3.2　評估3T3/Protein G及3T3/Poly-protein G抓取抗體之能力.....................19
3.3　3T3/protein-G或3T3/poly-protein-G所抓取之抗體，仍保有其抗體抗原之結合專一性...................................................................................................19
3.4　比較抗體固定於protein G cell-based ELISA平臺、poly-protein G cell-based ELISA平臺或傳統antibody based ELISA平臺之效率....................................20
3.5　比較poly-protein G cell based ELISA、protein G cell based ELISA與傳統antibody based ELISA其抗體偵測敏感度......................................................21
3.6 驗證poly-protein G cell based ELISA plate可應用於三明治酵素連結免疫吸附法( sandwich ELISA)並與傳統ELISA plate比較對於不同種類之PEG修飾物(PEG2K-Lipo-dox或PEG10K)之偵測敏感性...................................................22
3.7 評估傳統 ELISA plate以不同濃度之capture antibody偵測PEG10K 或PEG2K-Lipo-dox之偵測效率........................................................................23
3.8 比較poly-protein G cell based ELISA plate與傳統ELISA plate比較對於PEG修飾蛋白藥物 (PEG-alpha interferon 2α ; PEGASYS)之偵測敏感性................24
3.9 5 % FBS (Fetal bovine serum)可將未承載抗體之poly-protein G完全封閉..............................................................................................................25
第四章 討論
4.1 Poly-protein G cell based ELISA平臺較傳統ELISA平臺具備更的抗體承載量及偵測敏感性.........................................................................................27
4.2 何謂Protein G，及Protein G目前於各領域中之應用；並且應用於本 Poly- protein G cell based ELISA平臺之創新方法..........................................28
4.3 即使為未經純化之Capture antibody仍可直接應用於本Poly-protein G cell based ELISA平臺.......................................................................................28
4.4 Poly-protein G cell based ELISA平臺優於目前提高傳統ELISA平臺偵測效率之方法之................................................................................................29
4.5 新一代Poly-protein G cell based ELISA平臺可完全解決Antibody expressing cell based ELISA plate所面臨之問題............................................................30
第五章 結論
5.1 Poly-protein G cell based ELISA平臺之價值..........................................32
第六章 圖表 .................................................................................................34
第七章 參考資料 .........................................................................................44

1.Hornbeck P. Enzyme-linked immunosorbent assays. Current protocols in immunology / edited by John E Coligan [et al] 2001;Chapter 2:Unit 2.1.
2.Ayebazibwe C, Mwiine FN, Balinda SN, Tjornehoj K, Alexandersen S. Application of the Ceditest(R) FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 2012;24:270-6.
3.Huo SM, Yang H, Deng AP. Development and validation of a highly sensitive ELISA for the determination of pharmaceutical indomethacin in water samples. Talanta 2007;73:380-6.
4.Ravan H, Yazdanparast R. Loop region-specific oligonucleotide probes for loop-mediated isothermal amplification-enzyme-linked immunosorbent assay truly minimize the instrument needed for detection process. Analytical biochemistry 2013;439:102-8.
5.Yu FY, Gribas AV, Vdovenko MM, Sakharov IY. Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin B1 in food products. Talanta 2013;107:25-9.
6.Wang Y, Wei D, Yang H, et al. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples. Talanta 2009;77:1783-9.
7.Wang Y, Yang H, Pschenitza M, et al. Highly sensitive and specific determination of mercury(II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody. Analytical and bioanalytical chemistry 2012;403:2519-28.
8.Grothaus GD, Bandla M, Currier T, et al. Immunoassay as an analytical tool in agricultural biotechnology. Journal of AOAC International 2006;89:913-28.
9.Cao B, He G, Yang H, Chang H, Li S, Deng A. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Talanta 2013;115:624-30.
10.Han KC, Yang EG, Ahn DR. Elongated oligonucleotide-linked immunosorbent assay for sensitive detection of a biomarker in a microwell plate-based platform. Biosensors & bioelectronics 2013;50:421-4.
11.Wang G, Huang H, Zhang G, Zhang X, Fang B, Wang L. Dual amplification strategy for the fabrication of highly sensitive interleukin-6 amperometric immunosensor based on poly-dopamine. Langmuir : the ACS journal of surfaces and colloids 2011;27:1224-31.
12.Thaxton CS, Elghanian R, Thomas AD, et al. Nanoparticle-based bio-barcode assay redefines "undetectable" PSA and biochemical recurrence after radical prostatectomy. Proceedings of the National Academy of Sciences of the United States of America 2009;106:18437-42.
13.Butler JE, Ni L, Nessler R, et al. The physical and functional behavior of capture antibodies adsorbed on polystyrene. Journal of immunological methods 1992;150:77-90.
14.Kogot JM, Sarkes DA, Val-Addo I, Pellegrino PM, Stratis-Cullum DN. Increased affinity and solubility of peptides used for direct peptide ELISA on polystyrene surfaces through fusion with a polystyrene-binding peptide tag. BioTechniques 2012;52:95-102.
15.Chuang KH, Tzou SC, Cheng TC, et al. Measurement of poly(ethylene glycol) by cell-based anti-poly(ethylene glycol) ELISA. Analytical chemistry 2010;82:2355-62.
16.Olsson A, Eliasson M, Guss B, et al. Structure and evolution of the repetitive gene encoding streptococcal protein G. European journal of biochemistry / FEBS 1987;168:319-24.
17.Sjobring U, Bjorck L, Kastern W. Streptococcal protein G. Gene structure and protein binding properties. The Journal of biological chemistry 1991;266:399-405.
18.Omasa T, Onitsuka M, Kim WD. Cell engineering and cultivation of chinese hamster ovary (CHO) cells. Current pharmaceutical biotechnology 2010;11:233-40.
19.Aricescu AR, Owens RJ. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Current opinion in structural biology 2013;23:345-56.
20.Vink T, Oudshoorn-Dickmann M, Roza M, Reitsma JJ, de Jong RN. A simple, robust and highly efficient transient expression system for producing antibodies. Methods (San Diego, Calif) 2014;65:5-10.
21.Sule SV, Cheung JK, Antochshuk V, et al. Solution pH that minimizes self-association of three monoclonal antibodies is strongly dependent on ionic strength. Molecular pharmaceutics 2012;9:744-51.
22.Arosio P, Rima S, Morbidelli M. Aggregation mechanism of an IgG2 and two IgG1 monoclonal antibodies at low pH: from oligomers to larger aggregates. Pharmaceutical research 2013;30:641-54.
23.Li CH, Narhi LO, Wen J, et al. Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc. Biochemistry 2012;51:10056-65.
24.Han J, Zhuo Y, Chai Y, et al. Ultrasensitive electrochemical strategy for trace detection of APE-1 via triple signal amplification strategy. Biosensors & bioelectronics 2013;41:116-22.
25.Yuan Y, He H, Lee LJ. Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay. Biotechnology and bioengineering 2009;102:891-901.
26.Lin YH, Chen YJ, Lai CS, et al. A negative-pressure-driven microfluidic chip for the rapid detection of a bladder cancer biomarker in urine using bead-based enzyme-linked immunosorbent assay. Biomicrofluidics 2013;7:24103.
27.Hagan S, Tomlinson A. Tear fluid biomarker profiling: a review of multiplex bead analysis. The ocular surface 2013;11:219-35.