Antibody microarray-based profiling of complex specimens:: systematic evaluation of labeling strategies
Antibody microarray-based profiling of complex specimens:: systematic evaluation of labeling strategies
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DOI：
10.1002/pmic.200600762
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发表时间：
2007-06-01
期刊：
影响因子：
3.4
通讯作者：
Syagailo, Yana V.
中科院分区：
文献类型：
--
作者：
Kusnezow, Wlad;Banzon, Virryan;Syagailo, Yana V.
抗体微阵列在检测复杂样本中低丰度蛋白质方面的成功往往有限。信号放大系统改善了这种情况，但仍然相当费力和昂贵。然而，灵敏度问题更有可能是如前所述的动态适当的微阵列设计问题。因此，在这项研究中，我们重新检查了简单和廉价的检测方法对高灵敏抗体微阵列分析的适用性。用N-羟基琥珀酰亚胺酯(NHS)和通用连接系统(ULS)标记的荧光素和生物素分别作为随后与抗荧光素和抗生物素检测的标记，以及荧光染料用于血浆分析。分析并优化了影响芯片检测性能的参数，如标记条件、孵育时间、抗荧光素和抗生物素的浓度以及蛋白质标记的程度。间接检测策略，无论是基于NHS-或ULS-化学，都远远优于直接荧光标记，并能够以数十倍的信噪比检测到低丰度的细胞因子。最后，特别灵敏的检测化学被应用于监测刺激的外周T细胞的细胞因子的产生。微阵列数据与ELISA法和Luminex法测得的定量细胞因子水平一致，表明所建立的微阵列方法具有类似的可靠性和毫微摩尔范围敏感性。
Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.