2-ROUND ENZYMATIC AMPLIFICATION COMBINED WITH TIME-RESOLVED FLUOROMETRY OF TB3+ CHELATES FOR ENHANCED SENSITIVITY IN DNA HYBRIDIZATION ASSAYS

Microtiter well-based DNA hybridization assays are developed in which two rounds of enzymatic amplification are combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity. The target DNA is immobilized on the wells (through digoxigenin/antidigoxigenin inter action) and then hybridized with a biotinylated oligonucleotide probe. The hybrids are reacted with a streptavidin-horseradish peroxidase co njugate. Peroxidase catalyzes the oxidation of biotinylated tyramine b y hydrogen peroxide, resulting in the attachment of multiple biotin mo ieties to the solid phase. Alkaline phosphatase-labeled streptavidin i s then allowed to bind to the immobilized biotins. The activity of alk aline phosphatase is measured by using the phosphate ester of 5'-fluor osalicylate as a substrate. The fluorosalicylate produced forms a fluo rescent complex with Tb3+, which is measured by time-resolved fluorome try. We observed a 30-fold improvement of the signal and a 10-times en hancement of the singal-to-background ratio compared to the assay that uses a single round of enzymatic amplification (only alkaline phospha tase). The CV was in the range of 11.2-14.4%.