運用嶄新抗體固著策略發展及驗證新式抗體微晶片平台;Application of an Innovative Immobilization Strategy to the Development and Validation of a New Multiplexed Antibody Microarrays Platform

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Title:	運用嶄新抗體固著策略發展及驗證新式抗體微晶片平台;Application of an Innovative Immobilization Strategy to the Development and Validation of a New Multiplexed Antibody Microarrays Platform
Authors:	郭書麟;Guo,Shu-Lin
Contributors:	系統生物與生物資訊研究所
Keywords:	多孔位;多重分析;定量分析;疼痛相關訊息分子;蛋白質G;陣列式免疫分析法;array-based immunoassay;multiplexed;multi-well;pain-related message molecule;protein G;quantitation
Date:	2012-12-31
Issue Date:	2013-03-25 16:05:12 (UTC+8)
Publisher:	國立中央大學
Abstract:	為了打造因特殊研究需求所構建的，而進行相關蛋白質固著晶片表面效率之改進策略，則一直是系統生物學家非常興趣的一個課題。相較於核酸分子，蛋白質本身的功能是和它特有的三度空間結構緊密結合，當離開原本合宜的化學及物理環境，蛋白質就容易造成去活化質變的結果，自然地在製造抗體微陣列晶片就是個關鍵。因此，抗體微陣列晶片表面的選擇，必須因應不同抗體的特性而採取不同的考量；相對地，對於核酸微陣列製備過程不是問題。想進一步提昇微陣列晶片系統表現，其重點在於如何將抗體固著於晶片表面而不影響其構形及方向性。因為一個有效的固著方法必須適用於各種不同的抗體分子，當抗體固著於晶片表面時，可以不減損其活性，又可以保留與抗原結合之能力，乃是最佳選擇。這樣的解決方案於實際運用上，不僅應有再現性高，且有助於高通量自動化之數據產出，完整保留抗體結合能力及有效降低非特異性之鍵結等優點。在本研究中，我們提出了一個創新的抗體固著晶片的方案，利用蛋白質G鍵結於醛基晶片上，完全達成上述要求。為了同時顧及抗體活性及提昇微陣列反應，蛋白質G可以於攝氏4度下，相較於傳統方法，於較短時間內有效連結抗體及晶片表面，同時維持抗體排列方向一致性。我們以六種疼痛訊息傳遞分子為例進行抗體微陣列構建。相較於舊有抗體固著方式，蛋白質G的介入讓抗體固定於晶片時間，由原需隔夜作用有效縮短至只有二小時，大大提高實驗效率。同時，這個改良也讓抗體固著能力提昇，單位偵測密度增加，有利於高通量實驗之進行。所以，這個新的抗體微陣列平台，可以有效地觀察疼痛訊息傳遞分子於樣本中濃度變化。另外，本研究也提供實際實驗觀察，證實了蛋白質G能讓抗體微陣列分析敏感度改善、有效降低最低可偵測濃度及大幅提昇訊噪比。同時我們也進行了品管測試：首先，六種疼痛訊息傳遞分子平均有效降低最低可偵測濃度可以敏感至100 pg/ml，這樣的敏感度已優於許多同類型的檢驗方式；而在特異性方面，晶片的交叉反應測試上，相較於普通晶片，明顯改善其表現平均達50倍。總結以上結果，本研究以蛋白質G鍵結於醛基晶片表面的解決方案的確是高敏感性的檢驗方式，而且能有效縮短製備時間的創新作法。事實上，我們確實克服了幾項在發展抗體微陣列之可能會遭遇的困難，包括：以較短的抗體固著制備時間減少抗體變性的可能性；不需對抗體進行事前的化學修飾，可以完全保留對抗原的結合度及有效提升晶片的敏感度；更穩定的晶片固著能力可以提供晶片的再現性；以及一致性的抗體排列將有助於改善信噪比及減少非特異性結合的可能性。我們在小規模的實驗中證實了此新方法的可行性，這也為大規模應用奠定基礎。也相信以我們研發的平台上可以進一步發展出高敏感度的篩檢工具，尤其是可以有效地運用在研究生物標記、新藥標的開發，進而在研究上發展開拓其應有創新的價值。The construction of an antibody microarray for one specific target, with improved methods of protein immobilization on solid matrix, is always of great interest to the field of systems biology. Unlike DNA or RNA, proteins have unique 3D structures that are critical to their functions, but have the tendency of denaturing quickly after leaving their natural chemical and physical environments, a tendency that poses a serious challenge to making antibody microarrays viable. Consequently, the selection of suitable surface chemistry is requirement unique to antibody microarrays; it is not needed for, say, DNA microarrays. For enhancing the performance, how antibodies are captured and fixed on the slide surface plays an increasingly vital role in maintaining the conformation and orientation of the antibody. For an attachment method to be effective, it is necessary that it is applicable to a wide range of antibodies and, when the antibodies are bound to the surface of solid substrates, their activities and binding capacity are preserved. Immobilization methods using efficient surface chemistry should be reliable, applicable to antibodies with universal properties, amenable to high-throughput automation, and it should fully preserve the binding capabilities of the antibodies – through maintenance of the correct orientation of antibody epitopes – and minimize nonspecific binding.We present a novel immobilization method to capture antibodies that uses Protein G coating on aldehyde-derivatized slide. To maintain the antibody activity and enhance performance of array-based immunoassays, protein G was used to allow a shorter duration of immunoglobulin G immobilization at 4 °C, with the antibody placed in the appropriate orientation with uniform face-out epitopes. The multiplexed detection of six pain-related message molecules (PRMMs) was used as examples for the development of array-based immunoassays: substance P, calcitonin gene-related peptide, nerve growth factor, brain-derived neurotrophic factor, tumor necrosis factor-α, and β-endorphin. Compared to non-protein G immunoassays, protein G shortened the antibody immobilization time at 4 °C from overnight to 2 hours. It provides effective high-density protein immobilization without activity loss or incorrect orientation of the capture antibody. This new platform of antibody microarray is a useful tool for analysis of PRMMs as demonstrated in our experimental results with fluorescence immunoassay methods. A mechanism of protein binding to solid surface coated with Protein G that results in improved detection sensitivity, excellently low limit of detection (LOD), and remarkably high signal-to-noise (SN) ratio are provided. In our array validation, the LOD of six PRMMs were sensitive to average 100 pg/ml,ivbetter than other proteomic tools. Our method exhibited excellent specificity, the cross-reactivity was observed to have contrast 50-times seen typically in conventional array surface.In conclusion, our experimental results suggested that Protein G, a novel linker molecule, was an efficient antibody immobilizer, and that Protein G-coated method was a highly sensitive antibody microarray. Key obstacles overcame and achievement obtained in this work include: prevention of antibody denaturation by shortened immobilization time; full preservation of antibody’s binding capacity and effective increase of array sensitivity by absence of extraneous modification tag on antibody; good reproducibility assured by more stable fixation of antibody on surface; increase of signal-to-noise ratio and reduction of non-specific binding by uniform arrangement of capture antibody. We demonstrated the applicability for a small-scale project, and believe it forms the foundation large-scale scale-up. We believe our method can be a useful tool for the development of a high detection sensitivity, antigen-antibody interaction, microarray-based screening system for new drug target discovery and biomarker assays.
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