A Laboratory-Based Antibody Array for Profiling Cytokine Expression

目的：基因的表現並不能與數以百萬計的蛋白質表現畫上等號，因而也帶動蛋白質體學的研究，尤其是抗體微陣列之發展。雖然目前已有許多蛋白質晶片發展出來，卻由於蛋白質本身相較於DNA的不穩定性，以及市場的需求度，限制了蛋白質晶片的普及。為了配合實驗室研究的需求，我們嘗試利用實驗室自製之抗體微陣列，偵測部分細胞激素的表現。方法：我們利用點陣系統(SpotArray(上標 TM) 24)將不同細胞激素之單株抗體以四重復方式點在聚酯纖維玻片上(FAST(上標 TM) Slides)製成抗體微陣列，並存放於-20℃備用。結果：在控制組的測試中，自製晶片的敏感度可以到達100 pg/ml以上；此外，對於細胞在缺氧狀況下或是LPS刺激下所產生到培養液中之細胞激素的變化，都能偵測得到，而相似的結果在我們將自製晶片儲存於-20℃六個月後仍能重複觀察到。結論：這表示我們自製的抗體微陣列，能夠符合一般實驗室研究的需求。分析工具的發展，無疑是朝向分析速度快、結果精準及使用便利等方向來發展，因此若能廣泛利用自製之抗體微陣列，相信對於實驗室研究的發展定能有相當的助益，而且更能符合各實驗室需求。

關鍵字

蛋白質體學；蛋白質微陣列；細胞激素；多醣脂蛋白；局部缺血

並列摘要

Purpose: Despite the expansion in proteomics research, the instability and complexity of proteins limit the feasibility of adopting antibody arrays in laboratory studies. We developed a simple and rapid antibody array-based method to characterize cytokine expression and evaluated its validity and accuracy. Method: We spotted monoclonal antibodies against various cytokines onto a nitrocellulose slide (FAST(superscript TM) Slides) in tetrad with a microarray spotting system (SpotArray(superscript TM) 24) and stored the slides at -20°C until use, In addition, we subjected cells to lipopolysaccharide (LPS) and shear stress treatment to alter the cytokine secretion profiles and validated the antibody array method using reverse transcription-polymerase chain reaction (RT-PCR). Results: The sensitivity of the antibody array test was higher than 100 pg/ml. Variations in cytokine secretion due to LPS and shear stress-treated Raw 264.7 cells were detected by the array. The cytokine mRNA levels measured by the array were confirmed by RT-PCR analysis. The procedures took approximately 90 minutes. The antibody array reproduced similar results after being stored at -20°C for 6 months. Conclusion: The laboratory-based antibody array is efficient, accurate, and feasible for profiling cytokine expression.

並列關鍵字

proteomics ； protein array ； cytokines ； LPS ； ischemia

國際替代計量

A Laboratory-Based Antibody Array for Profiling Cytokine Expression

全文下載

主題瀏覽