Label-free detection of proteins in crude cell lysate with antibody arrays by a surface plasmon resonance imaging technique
Label-free detection of proteins in crude cell lysate with antibody arrays by a surface plasmon resonance imaging technique
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DOI：
10.1021/ac050884a
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发表时间：
2005-11-15
影响因子：
7.4
通讯作者：
Koga, H
中科院分区：
文献类型：
--
作者：
Kyo, M;Usui-Aoki, K;Koga, H
我们建立了一种使用抗体阵列和表面等离子共振 (SPR) 成像测量粗细胞裂解液中蛋白质的无标记方法。根据裂解液的折射率调整电泳缓冲液的折射率，以克服体积效应。研究了所制造阵列的化学成分，以减少阵列表面上的非特异性吸附。我们发现聚乙二醇部分的亲水性和表面较低的静电荷提供了抗原-抗体相互作用的特异性测量。我们通过测量小鼠大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来验证该系统。抗体阵列的检测限与粗细胞裂解液中的 30 ng/mL 相似，与之前的 SPR 研究的检测限相同。该系统能够以最小的样品体积（类似于 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。
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 similar to 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 (similar to 200 mu L). 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.