SANDWICH-TYPE DEOXYRIBONUCLEIC-ACID HYBRIDIZATION ASSAYS BASED ON ENZYME-AMPLIFIED TIME-RESOLVED FLUOROMETRY

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates, Th e target DNA was hybridized with two adjacent and non-overlapping olig onucleotide probes, one oligonucleotide serving as the capture probe a nd the other as the detection probe, Two ligand-specific binding prote in 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 c atalytic hydrolysis of the substrate produces diflunisal which forms t ernary fluorescent complex with Tb3+-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 hy brids through biotin-streptavidin and detection via digoxigenin-anti-d igoxigenin offer 2-3 times higher sensitivity than the reverse configu ration, The highest sensitivity was achieved with enzymatic labeling o f capture and detection probes at the 3' end. A signal-to-background r atio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.