Kyo Motoki, Usui-Aoki Kazue, Koga Hisashi
Biotechnology Frontier Project, Toyobo Co., Ltd., Tsuruga, Fukui, Japan.
Anal Chem. 2005 Nov 15;77(22):7115-21. doi: 10.1021/ac050884a.
We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.
我们建立了一种使用抗体阵列和表面等离子体共振（SPR）成像技术在粗细胞裂解物中测量蛋白质的无标记方法。通过调整运行缓冲液与裂解物的折射率来克服体积效应。研究了所制备阵列的化学性质，以减少阵列表面的非特异性吸附。我们发现聚乙二醇部分的亲水性和表面较低的静电荷能够提供对抗抗原-抗体相互作用的特异性测量。我们通过测量小鼠脑中八种蛋白质的表达并将结果与传统蛋白质印迹法的结果进行比较，验证了该系统。抗体阵列在粗细胞裂解物中的检测限约为30 ng/mL，与之前的SPR研究结果处于同一水平。该系统能够以最少的样品体积（约200 μL）对丰富的蛋白质进行快速、无标记和高通量分析。预计我们的SPR抗体阵列将适用于细胞裂解物的直接蛋白质表达谱分析，以及细胞表型分析、食品分析、新生物标志物的发现和免疫疾病诊断。
