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The sensitivity of aequorin-based bioluminometric hybridization assays was enhanced by introducing, enzymically, multiple aequorin labels per DNA hybrid. The target DNA was hybridized in microtiter wells with an immobilized capture probe and a digoxigenin-labeled detection probe. The hybrids were reacted with an anti-digoxigenin antibody conjugated to horseradish peroxidase. Peroxidase catalyzed the oxidation of digoxigenin-tyramine by hydrogen peroxide, resulting in the attachment of multiple digoxigenin moieties to the solid phase. Aequorin-labeled anti-digoxigenin antibody was then allowed to bind to the immobilized digoxigenins. The bound aequorin was determined by its characteristic Ca2+-triggered bioluminescence. As low as 20 fmol/L (1 amol/ well) target DNA was detected with a signal-to-background ratio of 2.7. A hybridization assay that used only aequorin-labeled anti-digoxigenin antibody without the peroxidase amplification step gave a signal-to-background ratio of 2 for 160 fmol/L target DNA. The signal enhancement of the amplified assay was in the range of 14-38 times. The analytical range of the amplified assay extended up to 2600 fmol/L. The CVs were in the range of 5.5-7.3%.

Two cDNAs encoding firefly luciferase (FLuc) and Renilla luciferase (RLuc) were used as labels for the development of a microtiter well-based expression hybridization assay that allows simultaneous determination of two target DNA sequences. The target DNAs were denatured and hybridized with specific capture and detection probes. One detection probe was biotinylated while the other was tailed with poly(dT). The hybrids were reacted with a streptavidin−FLuc DNA complex and a poly(dA)-tailed RLuc DNA, respectively. Subsequently, the cDNA labels were expressed in vitro simultaneously and independently in the same transcription/translation reaction mixture. The activities of generated firefly and Renilla luciferases were co-determined in the same sample based on the differential requirements of their characteristic bioluminescent reactions for magnesium ions.