Group of Mechanism and Regulation of DNA Repair and IMPACT Platform, UFIP UMR CNRS 6286/University of Nantes, Nantes, France.
Laboratory of Nano-Bioengineering, National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow, Russian Federation.
Methods Mol Biol. 2020;2135:259-273. doi: 10.1007/978-1-0716-0463-2_16.
Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.
抗体微阵列已成为多重免疫分析技术中的强大工具。微阵列技术的优势在于有可能在单个样本中快速分析多个靶标，具有高灵敏度，这使它们成为高通量筛选的理想选择。通常，这些微阵列包含生物识别分子，例如全长抗体、抗原结合片段和单域抗体，以及用于检测的标记物。有机荧光团是最受欢迎的标记物，但由于光降解，它们的灵敏度和稳定性较低。在这里，我们描述了一种使用高荧光半导体纳米晶体或量子点 (QD) 作为荧光信号源制造抗体微阵列的方案，这可能会显著改善微阵列的性能，包括其灵敏度和特异性。我们的分析物检测方法基于使用链霉亲和素-生物素来评估和监测荧光信号，而不是直接标记样品，这有助于提高结果的重现性和微阵列的灵敏度。已测试开发的抗体微阵列用于检测神经胶质细胞系中的 DNA-PKcs 以及用不同蛋白激酶抑制剂在 HeLa 细胞中测量喜树碱诱导的 DNA 损伤引起的细胞蛋白磷酸化变化。
