Chiu N H, Christopoulos T K, Peltier J
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Analyst. 1998 Jun;123(6):1315-9. doi: 10.1039/a707528f.
We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.
我们报道了基于微孔板的夹心型DNA杂交分析方法，该方法采用铽（Tb3+）螯合物的酶放大时间分辨荧光法。靶DNA与两个相邻且不重叠的寡核苷酸探针杂交，一个寡核苷酸用作捕获探针，另一个用作检测探针。交替使用两对配体特异性结合蛋白将杂交体捕获到固相并进行检测，即生物素-链霉亲和素和地高辛配基-抗地高辛配基相互作用。在这两种情况下，碱性磷酸酶用作报告分子，二氟尼柳磷酸用作底物。底物的催化水解产生二氟尼柳，其与Tb(3+)-EDTA形成三元荧光复合物。此外，我们研究了探针标记方法和标记位置对分析灵敏度的影响。数据表明，通过生物素-链霉亲和素捕获杂交体并通过地高辛配基-抗地高辛配基进行检测，其灵敏度比反向配置高2-3倍。在捕获探针和检测探针的3'端进行酶标记可实现最高灵敏度。对于0.2 fmol的靶DNA，信噪比达到4。相对标准偏差优于4%。
