Optimization of a microarray sandwich-ELISA against hINF-gamma on a modified nitrocellulose membrane.
Optimization of a microarray sandwich-ELISA against hINF-gamma on a modified nitrocellulose membrane.
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在改性硝酸纤维素膜上针对 hINF-gamma 的微阵列夹心 ELISA 的优化。
DOI：
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
T. Scheper
中科院分区：
文献类型：
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作者：
M. Reck;F. Stahl;J. Walter;M. Hollas;D. Melzner;T. Scheper
高特异性和高灵敏度的ELISA（酶联免疫吸附测定）技术通常是用于免疫诊断的最常用方法。结合蛋白质微阵列及其允许并行执行数千个实验的能力，给出了一种具有减少时间、分析物和试剂消耗的全球分析方法的有前途的工具。本研究建立了一种基于蛋白芯片的人干扰素-γ（hINF-γ）的ELISA检测方法。考虑到表面化学的巨大重要性，测试并优化了一种新的黑色硝化纤维素基质作为微阵列基底，该基质产生非常高的信噪比（SNR）和非常低的自发荧光。通过与不同的市售系统进行比较，验证了该系统的适用性。实验结果表明，基于微阵列的ELISA是更快，更容易执行，并显示出较低的检测限（LOD）比可比系统在96孔板。带有捕获抗体的点样载玻片可储存长达1个月，信号强度无显著损失。具有固定化His标记的限制性酶EcoRV和抗His抗体的第二模型系统同时显示黑色硝酸纤维素膜的良好适用性和对ELISA的无交叉反应性。
The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray-based sandwich-ELISA for human interferon-gamma (hINF-gamma) is established. In consideration of the immense importance of the surface chemistry, a new black nitrocellulose matrix that generates very high signal-to-noise ratios (SNR) and a very low autofluorescence was tested and optimized as microarray substrate. A validation of the applicability of the system was performed with a comparison to different commercially available systems. Experimental results show that the microarray-based ELISA is faster and easier to perform and shows a lower limit of detection (LOD) than a comparable system in a 96-well plate. The spotted slides with the capture antibody can be stored up to 1 month with no significant loss of signal intensity. A second model system with immobilized His-tagged restriction enzyme EcoRV and an anti-His antibody shows in coincidence the good applicability of the black nitrocellulose membrane and no cross-reactivity toward the ELISA.