開發創新的poly-protein G表現細胞以提升酵素免疫吸附測定法(ELISA)之抗體偵測效率

Development of a novel poly-protein G expressing cell to enhance detection sensitivity of antibody based enzyme-linked immunosorbent assay(ELISA)

陳易柔(Yi-Jou Chen)

指導教授 ： 莊國祥

臺北醫學大學/藥學院/生藥學研究所/碩士(2014年)

摘要

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。

關鍵字

protein G ； poly-protein G表現細胞 ； 酵素免疫分析法 ； ELISA

並列摘要

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.

並列關鍵字

protein G ； poly-protein G expressing cells ； enzyme linked immunosorbent assay ； ELISA

參考文獻

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.